Anti-c5 antibodies and uses thereof

ABSTRACT

The present invention provides monoclonal antibodies that bind to the complement factor 5 (C5) protein, and methods of use thereof. In various embodiments of the invention, the antibodies are fully human antibodies that bind to C5 protein. In some embodiments, the antibodies of the invention are useful for inhibiting or neutralizing C5 activity, thus providing a means of treating or preventing a C5-related disease or disorder in humans. In some embodiments, the invention provides for an anti-C5 antibody that has improved pharmacokinetic and pharmacodynamic properties, e.g., a half-life of more than 10 days.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/621,689 filed Jun. 13, 2017, which claims the benefit of priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Nos.62/349,705 filed Jun. 14, 2016, 62/405,561 filed Oct. 7, 2016, and62/422,107 filed Nov. 15, 2016, the disclosures of all of which arehereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention is related to antibodies and antigen-bindingfragments of antibodies that specifically bind to complement factor C5,and therapeutic and diagnostic methods of using those antibodies.

BACKGROUND OF THE INVENTION

The complement system is a group of plasma proteins that when activatedlead to target cell lysis and facilitate phagocytosis throughopsonization. Complement is activated through a series of proteolyticsteps by three major pathways: the classical pathway, which is typicallyactivated by immune-complexes, the alternative pathway that can beinduced by unprotected cell surfaces, and the mannose binding lectinpathway. All three pathways of complement cascade converge onproteolytic cleavage of complement component 5 (C5) protein. Cleavage ofcomplement component 5 (C5) results in the production of fragments C5aand C5b, a process that is critical during the activation of thecomplement cascade. C5a can generate pleiotropic physiological responsesthrough binding to its receptors (Monk et al 2007, Br. J. Pharmacol.152: 429-448). C5a is a potent pro-inflammatory mediator that induceschemotactic migration, enhances cell adhesion, stimulates the oxidativeburst, and induces the release of various inflammatory mediators such ashistamine or cytokines. C5b mediates the formation of themembrane-attack complex (MAC, or C5b-9) leading to cell lysis in thelate phases of the complement dependent cytotoxicity (CDC). Further, innucleated cells that are resistant to cytolysis by C5b-9, sublyticquantities of C5b-9 can cause cellular activation which results in cellproliferation, generation of pro-inflammatory mediators and productionof extracellular matrix.

Monoclonal antibodies to C5 are known in the art and have beendescribed, for example, in U.S. Pat. Nos. 9,206,251, 9,107,861,9,079,949, 9,051,365, 8,999,340, 8,883,158, 8,241,628, 7,999,081,7,432,356, 7,361,339, 7,279,158, 6,534,058, 6,355,245, 6,074,642,20160299305, 20160051673, 20160031975, 20150158936, 20140056888,20130022615, 20120308559, and in WO2015198243, WO2015134894,WO2015120130, EP2563813B1, EP2328616B1, and EP2061810B1.

Fully human antibodies that specifically bind to C5 protein with highaffinity and have improved pharmacokinetic properties could be importantin the prevention and treatment of various C5-associated diseases (e.g.,atypical hemolytic uremic syndrome).

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies and antigen-binding fragmentsthereof that specifically bind complement factor 5 (C5) protein. Theantibodies of the present invention are useful, inter alia, forinhibiting or neutralizing the activity of C5 protein. In certainembodiments, the antibodies are useful in preventing, treating orameliorating at least one symptom or indication of a C5-associateddisease or disorder in a subject. In certain embodiments, the antibodiesmay be administered prophylactically or therapeutically to a subjecthaving or at risk of having a C5-associated disease or disorder. Incertain embodiments, the anti-C5 antibodies are fully human antibodiesthat bind to C5 with high affinity and have improved pharmacokinetic(PK) and pharmacodynamic (PD) properties. Such high-affinity antibodieswith improved PK/PD can be used to provide superior efficacy, along withless frequent dosing in a subject with a C5-associated disease ordisorder.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to increase persistence in the host or to eliminateresidual effector functions (Reddy et al., 2000, J. Immunol.164:1925-1933). In certain embodiments, the antibodies may bebispecific.

In a first aspect, the present invention provides isolated recombinantmonoclonal antibodies or antigen-binding fragments thereof that bindspecifically to the C5 protein. In some embodiments, the antibodies arefully human monoclonal antibodies.

Exemplary anti-C5 antibodies of the present invention are listed inTables 1 and 2 herein. Table 1 sets forth the amino acid sequenceidentifiers of the heavy chain variable regions (HCVRs), light chainvariable regions (LCVRs), heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3), and light chain complementaritydetermining regions (LCDR1, LCDR2 and LCDR3) of exemplary anti-C5antibodies. Table 2 sets forth the nucleic acid sequence identifiers ofthe HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of theexemplary anti-C5 antibodies.

The present invention provides antibodies, or antigen-binding fragmentsthereof, comprising an HCVR comprising an amino acid sequence selectedfrom any of the HCVR amino acid sequences listed in Table 1, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an LCVR comprising an amino acid sequenceselected from any of the LCVR amino acid sequences listed in Table 1, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an HCVR and an LCVR amino acid sequencepair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listedin Table 1 paired with any of the LCVR amino acid sequences listed inTable 1. According to certain embodiments, the present inventionprovides antibodies, or antigen-binding fragments thereof, comprising anHCVR/LCVR amino acid sequence pair contained within any of the exemplaryanti-C5 antibodies listed in Table 1. In certain embodiments, theHCVR/LCVR amino acid sequence pair is selected from the group consistingof SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 98/114,122/106, 98/130, 138/106, 146/106, 122/130, 146/114, 146/130, 138/130,154/162, 170/178, 186/194, 202/210, 218/226, 234/242, 250/258, 266/258,274/282, 290/298, 306/314, 322/330, and 338/346. In certain embodiments,the HCVR/LCVR amino acid sequence pair is selected from one of SEQ IDNOs: 50/58 (e.g., H4H12161P), 98/106 (e.g., H4H12166P), 138/106 (e.g.,H4H12166P5), or 202/210 (e.g., H4H12170P). In certain embodiments, thepresent invention provides anti-C5 antibodies or antigen-bindingfragments thereof comprising a HCVR and a LCVR, said HCVR comprising anamino acid sequence listed in Table 1 having no more than five aminoacid substitutions, and said LCVR comprising an amino acid sequencelisted in Table 1 having no more than two amino acid substitutions. Forexample, the present invention provides anti-C5 antibodies orantigen-binding fragments thereof comprising a HCVR and a LCVR, saidHCVR comprising an amino acid sequence of SEQ ID NO: 98 having no morethan five amino acid substitutions, and said LCVR comprising an aminoacid sequence of SEQ ID NO: 106 having no more than two amino acidsubstitutions. In another embodiment, the present invention providesanti-C5 antibodies or antigen-binding fragments thereof comprising aHCVR and a LCVR, said HCVR comprising an amino acid sequence of SEQ IDNO: 98 having at least one amino acid substitution, and said LCVRcomprising an amino acid sequence of SEQ ID NO: 106 having one aminoacid substitution.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising anamino acid sequence selected from any of the HCDR1 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising anamino acid sequence selected from any of the HCDR2 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising anamino acid sequence selected from any of the HCDR3 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR1 (LCDR1) comprising anamino acid sequence selected from any of the LCDR1 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR2 (LCDR2) comprising anamino acid sequence selected from any of the LCDR2 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain CDR3 (LCDR3) comprising anamino acid sequence selected from any of the LCDR3 amino acid sequenceslisted in Table 1 or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequencepair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequenceslisted in Table 1 paired with any of the LCDR3 amino acid sequenceslisted in Table 1. According to certain embodiments, the presentinvention provides antibodies, or antigen-binding fragments thereof,comprising an HCDR3/LCDR3 amino acid sequence pair contained within anyof the exemplary anti-C5 antibodies listed in Table 1. In certainembodiments, the HCDR3/LCDR3 amino acid sequence pair is selected fromthe group consisting of SEQ ID NOs: 56/64 (e.g., H4H12161P), 104/112(e.g., H4H12166P), 144/112 (e.g., H4H12166P5), and 208/216 (e.g.,H4H12170P).

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a HCVR and a LCVR, said HCVR comprisingHCDR1 comprising an amino acid sequence differing from an amino acidsequence listed in Table 1 by 1 amino acid, HCDR2 comprising an aminoacid sequence differing from an amino acid sequence listed in Table 1 by1 amino acid, and HCDR3 comprising an amino acid sequence differing froman amino acid sequence listed in Table 1 by 1 amino acid. In certainembodiments, the present invention provides antibodies, orantigen-binding fragments thereof, comprising a HCVR and a LCVR, saidLCVR comprising LCDR1 comprising an amino acid sequence differing froman amino acid sequence listed in Table 1 by 1 amino acid, LCDR2comprising an amino acid sequence differing from an amino acid sequencelisted in Table 1 by 1 amino acid, and LCDR3 comprising an amino acidsequence differing from an amino acid sequence listed in Table 1 by 1amino acid. For example, the present invention provides anti-C5antibodies, or antigen-binding fragments thereof, comprising a HCVR anda LCVR, said HCVR comprising HCDR1 comprising an amino acid sequence ofSEQ ID NO: 100 or an amino acid sequence differing from SEQ ID NO: 100by 1 amino acid, HCDR2 comprising an amino acid sequence of SEQ ID NO:102 or an amino acid sequence differing from SEQ ID NO: 102 by 1 aminoacid, and HCDR3 comprising an amino acid sequence of SEQ ID NO: 104 oran amino acid sequence differing from SEQ ID NO: 104 by 1 amino acid. Inanother exemplary embodiment, the present invention provides antibodies,or antigen-binding fragments thereof, comprising a HCVR and a LCVR, saidLCVR comprising LCDR1 comprising an amino acid sequence of SEQ ID NO:108 or an amino acid sequence differing from SEQ ID NO: 108 by 1 aminoacid, LCDR2 comprising an amino acid sequence of SEQ ID NO: 110 or anamino acid sequence differing from SEQ ID NO: 110 by 1 amino acid, andLCDR3 comprising an amino acid sequence of SEQ ID NO: 112 or an aminoacid sequence differing from SEQ ID NO: 112 by 1 amino acid.

The present invention provides antibodies, or antigen-binding fragmentsthereof, comprising a heavy chain comprising an amino acid sequence ofSEQ ID NO: 353, or a substantially similar sequence thereof having atleast 80%, at least 85%, at least 90%, at least 95%, at least 98% or atleast 99% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a light chain comprising an amino acidsequence of SEQ ID NO: 354, or a substantially similar sequence thereofhaving at least 80%, at least 85%, at least 90%, at least 95%, at least98% or at least 99% sequence identity thereto.

In certain embodiments, the present invention provides antibodies, orantigen-binding fragments thereof, comprising a heavy chain comprisingan amino acid sequence of SEQ ID NO: 353, or a substantially similarsequence thereof having at least 80%, or at least 90% sequence identitythereto; and a light chain comprising an amino acid sequence of SEQ IDNO: 354, or a substantially similar sequence thereof having at least80%, or at least 90% sequence identity thereto.

The present invention also provides antibodies, or antigen-bindingfragments thereof, comprising a set of six CDRs (i.e.,HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of theexemplary anti-C5 antibodies listed in Table 1. In certain embodiments,the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set isselected from the group consisting of SEQ ID NOs: 52-54-56-60-62-64(e.g., H4H12161P), 100-102-104-108-110-112 (e.g., H4H12166P),140-142-144-108-110-112 (e.g., H4H12166P5), and 204-206-208-212-214-216(e.g., H4H12170P).

In a related embodiment, the present invention provides antibodies, orantigen-binding fragments thereof, comprising a set of six CDRs (i.e.,HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR aminoacid sequence pair as defined by any of the exemplary anti-C5 antibodieslisted in Table 1. For example, the present invention includesantibodies, or antigen-binding fragments thereof, comprising theHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set containedwithin an HCVR/LCVR amino acid sequence pair selected from the groupconsisting of SEQ ID NOs: 50/58 (e.g., H4H12161P), 98/106 (e.g.,H4H12166P), 138/106 (e.g., H4H12166P5), or 202/210 (e.g., H4H12170P).Methods and techniques for identifying CDRs within HCVR and LCVR aminoacid sequences are well known in the art and can be used to identifyCDRs within the specified HCVR and/or LCVR amino acid sequencesdisclosed herein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

In certain embodiments, the present invention includes an antibody orantigen-binding fragment thereof that binds specifically to C5, whereinthe antibody or antigen-binding fragment thereof comprises three heavychain complementarity determining regions (CDRs) (HCDR1, HCDR2 andHCDR3) contained within a heavy chain variable region (HCVR) and threelight chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chainvariable region (LCVR), wherein the HCVR comprises: (i) the amino acidsequence of SEQ ID NO: 98, (ii) an amino acid sequence having at least90% identity to SEQ ID NO: 98, (iii) an amino acid sequence having atleast 95% identity to SEQ ID NO: 98; or (iv) the amino acid sequence ofSEQ ID NO: 98 having no more than 5 amino acid substitutions; and theLCVR comprises: (i) the amino acid sequence of SEQ ID NO: 106, (ii) anamino acid sequence having at least 90% identity to SEQ ID NO: 106,(iii) an amino acid sequence having at least 95% identity to SEQ ID NO:106; or (iv) the amino acid sequence of SEQ ID NO: 106 having no morethan 5 amino acid substitutions.

The present invention includes anti-C5 antibodies having a modifiedglycosylation pattern. In some embodiments, modification to removeundesirable glycosylation sites may be useful, or an antibody lacking afucose moiety present on the oligosaccharide chain, for example, toincrease antibody dependent cellular cytotoxicity (ADCC) function (seeShield et al. (2002) JBC 277:26733). In other applications, modificationof galactosylation can be made in order to modify complement dependentcytotoxicity (CDC).

In certain embodiments, the present invention provides antibodies andantigen-binding fragments thereof that exhibit pH-dependent binding toC5. For example, the present invention includes antibodies andantigen-binding fragment thereof that bind 05 with higher affinity atneutral pH than at acidic pH (i.e., reduced binding at acidic pH).

In certain embodiments, the present invention provides antibodies andantigen-binding fragments that exhibit improved pharmacokinetic andpharmacodynamic properties, for example, the present invention providesanti-C5 antibodies that have extended serum half-life. In certainembodiments, the anti-C5 antibodies of the present invention have serumconcentration of more than 10 μg/mL through day 40 in C5-humanized mice.In certain embodiments, the anti-C5 antibodies of the present inventionblock CP- and AP hemolysis through day 35 upon administration toC5-humanized mice.

The present invention also provides for antibodies and antigen-bindingfragments thereof that compete for specific binding to C05 with anantibody or antigen-binding fragment thereof comprising the CDRs of aHCVR and the CDRs of a LCVR, wherein the HCVR and LCVR each has an aminoacid sequence selected from the HCVR and LCVR sequences listed in Table1.

The present invention also provides antibodies and antigen-bindingfragments thereof that cross-compete for binding to C05 with a referenceantibody or antigen-binding fragment thereof comprising the CDRs of aHCVR and the CDRs of a LCVR, wherein the HCVR and LCVR each has an aminoacid sequence selected from the HCVR and LCVR sequences listed in Table1.

The present invention also provides antibodies and antigen-bindingfragments thereof that bind to the same epitope as a reference antibodyor antigen-binding fragment thereof comprising the CDRs of a HCVR andthe CDRs of a LCVR, wherein the HCVR and LCVR each has an amino acidsequence selected from the HCVR and LCVR sequences listed in Table 1. Incertain embodiments, the present invention provides antibodies andantigen-binding fragments thereof that bind to the same epitope as areference antibody or antigen-binding fragment thereof comprising theCDRs of a HCVR and the CDRs of a LCVR, wherein the HCVR/LCVR amino acidsequence pair has SEQ ID NOs: 98/106.

The present invention also includes anti-C5 antibodies andantigen-binding fragments thereof that bind to one or more amino acidresidues comprised in the alpha chain and/or the beta chain of C5. Incertain embodiments, the present invention provides antibodies andantigen-binding fragments thereof that bind to one or more amino acidsin the alpha chain of C5 and one or more amino acids in the beta chainof C5. In certain embodiments, the present invention provides antibodiesand antigen-binding fragments thereof that bind to one or more aminoacids in the alpha and beta chains of C5, wherein the antibodies do notbind to the C5a anaphylatoxin domain. In certain embodiments, thepresent invention provides anti-C5 antibodies that interact with one ormore amino acids contained within human C5 (SEQ ID NO: 359). In certainembodiments, the present invention provides anti-C5 antibodies thatinteract with one or more amino acids contained within human C5 (SEQ IDNO: 359), wherein the antibodies do not bind to the C5a anaphylatoxindomain of 05. In certain embodiments, the present invention providesanti-C5 antibodies and antigen-binding fragments thereof that interactwith an amino acid sequence selected from the group consisting of (a)amino acids 591 to 599 of SEQ ID NO: 359; (b) amino acids 593 to 599 ofSEQ ID NO: 359; (c) amino acids 775 to 787 of SEQ ID NO: 359; (d) aminoacids 775 to 794 of SEQ ID NO: 359; and (e) amino acids 779 to 787 ofSEQ ID NO: 359. In certain embodiments, the present invention providesanti-C5 antibodies and antigen-binding fragments thereof that interactwith one or more amino acids contained within SEQ ID NO: 359, forexample, the present invention provides anti-C5 antibodies andantigen-binding fragments thereof that interact with at least 5 aminoacids, at least 10 amino acids, or at least 15 amino acids containedwithin SEQ ID NO: 361. In certain embodiments, the present inventionprovides anti-C5 antibodies and antigen-binding fragments thereof thatinteract with one or more amino acids contained within SEQ ID NO: 359,for example, the present invention provides anti-C5 antibodies andantigen-binding fragments thereof that interact with at least 5 aminoacids contained within SEQ ID NO: 360. In certain embodiments, thepresent invention provides anti-C5 antibodies and antigen-bindingfragments thereof that interact with at least 5 amino acids containedwithin SEQ ID NOs: 360 and 361. In certain embodiments, the presentinvention provides anti-C5 antibodies and antigen-binding fragmentsthereof that interact with the amino acid sequence of SEQ ID NO: 360(corresponding to amino acids 591 to 599 of SEQ ID NO: 359) and with theamino acid sequence of SEQ ID NO: 361 (corresponding to amino acids 775to 794 of SEQ ID NO: 359).

In some embodiments, the antibody or antigen binding fragment thereofmay bind specifically to C5 in an agonist manner, i.e., it may enhanceor stimulate C5 binding and/or activity; in other embodiments, theantibody may bind specifically to C5 in an antagonist manner, i.e., itmay block C5 binding and/or activity.

The present invention also provides isolated antibodies andantigen-binding fragments thereof that block C5 binding to C5convertase. In some embodiments, the antibody or antigen-bindingfragment thereof that blocks C5 binding to C5 convertase may bind to thesame epitope on C5 as C5 convertase or may bind to a different epitopeon C5 as C5 convertase. In some embodiments, the present inventionprovides antibodies or antigen-binding fragments thereof that block thebinding of C5 to monkey C5 convertase.

In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention are bispecific comprising a first bindingspecificity to a first epitope of C5 protein and a second bindingspecificity to a second epitope of C5 protein wherein the first andsecond epitopes are distinct and non-overlapping.

In certain embodiments, the antibodies and antigen-binding fragments ofthe present invention bind to C5a with an IC₅₀ of less than 0.5 nM. Incertain embodiments, the antibodies comprise an HCVR comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 290,306, 322, and 338. In certain embodiments, the antibodies comprise anLCVR comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 298, 314, 330, and 346.

In certain embodiments, the present invention provides an isolatedantibody or antigen-binding fragment thereof that has one or more of thefollowing characteristics: (a) is a fully human monoclonal antibody; (b)binds to human C5 with a dissociation constant (K_(D)) of less than 0.9nM at 25° C., as measured in a surface plasmon resonance assay; (c)binds to human C5 with a K_(D) of less than 0.3 nM at 37° C., asmeasured in a surface plasmon resonance assay; (d) binds to monkey C5with a K_(D) of less than 65 nM, as measured in a surface plasmonresonance assay; (e) binds to human C5 variant R885H (SEQ ID NO: 356)with a K_(D) of less than 0.5 nM, as measured in a surface plasmonresonance assay; (f) binds to human C5 variant R885C (SEQ ID NO: 357)with a K_(D) of less than 0.5 nM, as measured in a surface plasmonresonance assay; (g) blocks human C5-mediated classical pathway (CP)hemolysis by more than 95% and with IC₅₀ less than 6 nM, as measured ina CP hemolysis assay; (h) blocks human C5-mediated alternative pathway(AP) hemolysis by more than 70% and with IC₅₀ less than 165 nM, asmeasured in a AP hemolysis assay; (i) inhibits African green monkeyC5-mediated CP hemolysis with IC₅₀ less than 185 nM, as measured in a CPhemolysis assay; (j) inhibits African green monkey C5-mediated APhemolysis with IC₅₀ less than 235 nM, as measured in a AP hemolysisassay; (k) inhibits cynomolgus monkey C5-mediated CP hemolysis with IC₅₀less than 145 nM, as measured in a CP hemolysis assay; and (I) inhibitscynomolgus monkey C5-mediated AP hemolysis with IC₅₀ less than 30 nM, asmeasured in a AP hemolysis assay.

In certain embodiments, the present invention provides an isolatedrecombinant monoclonal anti-C5 antibody or antigen-binding fragmentthereof that has one or more of the following characteristics: (a)comprises a set of six CDRs comprising the amino acid sequences of SEQID NOs: 100-102-104-108-110-112; (b) binds to human C5 with adissociation constant (K_(D)) of less than 0.2 nM at 25° C., as measuredin a surface plasmon resonance assay; (c) binds to human C5 with a K_(D)of less than 0.3 nM at 37° C., as measured in a surface plasmonresonance assay; (d) binds to a human C5 variant (R885H) with a K_(D) ofless than 0.4 nM at 37° C., as measured in a surface plasmon resonanceassay; (e) inhibits classical pathway (CP)-mediated hemolysis of humanserum with an IC₅₀ of less than 3 nM; (f) inhibits alternative pathway(AP)-mediated hemolysis of human serum with an IC₅₀ of less than 27 nM;(g) inhibits CP-mediated hemolysis of monkey serum with an IC₅₀ of lessthan 21 nM; (g) inhibits AP-mediated hemolysis of monkey serum with anIC₅₀ of less than 10 nM; (h) has serum half-life (t_(1/2)) of more than10 days in C5-humanized mice; (i) has serum concentration of more than10 μg/mL through day 40 upon administering to C5-humanized mice; (j)blocks CP-mediated hemolysis through day 50 in C5-humanized mice; and(k) binds to one or more amino acids comprised in the alpha chain and/orthe beta chain of SEQ ID NO: 359, wherein the antibody does not bind tothe C5a anaphylatoxin domain of 05.

In a second aspect, the present invention provides nucleic acidmolecules encoding anti-C5 antibodies or portions thereof. For example,the present invention provides nucleic acid molecules encoding any ofthe HCVR amino acid sequences listed in Table 1; in certain embodimentsthe nucleic acid molecule comprises a polynucleotide sequence selectedfrom any of the HCVR nucleic acid sequences listed in Table 2, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anHCVR, wherein the HCVR comprises a set of three CDRs (i.e.,HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequenceset is as defined by any of the exemplary anti-C5 antibodies listed inTable 1.

The present invention also provides nucleic acid molecules encoding anLCVR, wherein the LCVR comprises a set of three CDRs (i.e.,LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequenceset is as defined by any of the exemplary anti-C5 antibodies listed inTable 1.

The present invention also provides nucleic acid molecules encoding bothan HCVR and an LCVR, wherein the HCVR comprises an amino acid sequenceof any of the HCVR amino acid sequences listed in Table 1, and whereinthe LCVR comprises an amino acid sequence of any of the LCVR amino acidsequences listed in Table 1. In certain embodiments, the nucleic acidmolecule comprises a polynucleotide sequence selected from any of theHCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto, and a polynucleotide sequenceselected from any of the LCVR nucleic acid sequences listed in Table 1,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity thereto. Incertain embodiments according to this aspect of the invention, thenucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR andLCVR are both derived from the same anti-C5 antibody listed in Table 1.

In a related aspect, the present invention provides recombinantexpression vectors capable of expressing a polypeptide comprising aheavy or light chain variable region of an anti-C5 antibody. Forexample, the present invention includes recombinant expression vectorscomprising any of the nucleic acid molecules mentioned above, i.e.,nucleic acid molecules encoding any of the HCVR, LCVR, and/or CDRsequences as set forth in Table 2. Also included within the scope of thepresent invention are host cells into which such vectors have beenintroduced, as well as methods of producing the antibodies or portionsthereof by culturing the host cells under conditions permittingproduction of the antibodies or antibody fragments, and recovering theantibodies and antibody fragments so produced.

In a third aspect, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of at least onerecombinant monoclonal antibody or antigen-binding fragment thereofwhich specifically binds C5 and a pharmaceutically acceptable carrier.In a related aspect, the invention features a composition which is acombination of an anti-C5 antibody and a second therapeutic agent. Inone embodiment, the second therapeutic agent is any agent that isadvantageously combined with an anti-C5 antibody. Exemplary agents thatmay be advantageously combined with an anti-C5 antibody include, withoutlimitation, other agents that bind and/or inhibit C5 activity (includingother antibodies or antigen-binding fragments thereof, etc.) and/oragents which do not directly bind C5 but nonetheless treat or ameliorateat least one symptom or indication of a C5-associated disease ordisorder. Additional combination therapies and co-formulations involvingthe anti-C5 antibodies of the present invention are disclosed elsewhereherein.

In a fourth aspect, the invention provides therapeutic methods fortreating a disease or disorder associated with C5 in a subject using ananti-C5 antibody or antigen-binding portion of an antibody of theinvention, wherein the therapeutic methods comprise administering atherapeutically effective amount of a pharmaceutical compositioncomprising an antibody or antigen-binding fragment of an antibody of theinvention to the subject in need thereof. The disorder treated is anydisease or condition which is improved, ameliorated, inhibited orprevented by inhibition of C5 activity. In certain embodiments, theinvention provides methods to prevent, treat or ameliorate at least onesymptom of atypical hemolytic uremic syndrome (aHUS), the methodcomprising administering a therapeutically effective amount of ananti-C5 antibody or antigen-binding fragment thereof of the invention toa subject in need thereof. In some embodiments, the present inventionprovides methods to ameliorate or reduce the severity of at least onesymptom or indication of paroxysmal nocturnal hemoglobinuria (PNH) in asubject by administering an anti-C5 antibody of the invention. In someembodiments, the antibody or antigen-binding fragment thereof may beadministered prophylactically or therapeutically to a subject having orat risk of having a C5-associated disease or disorder. In certainembodiments, the antibody or antigen-binding fragment thereof theinvention is administered in combination with a second therapeutic agentto the subject in need thereof. The second therapeutic agent may beselected from the group consisting of an anti-inflammatory drug (such ascorticosteroids, and non-steroidal anti-inflammatory drugs), a differentantibody to C5 protein, a dietary supplement such as anti-oxidants andany other drug or therapy known in the art. In certain embodiments, thesecond therapeutic agent may be an agent that helps to counteract orreduce any possible side effect(s) associated with an antibody orantigen-binding fragment thereof of the invention, if such sideeffect(s) should occur. The antibody or fragment thereof may beadministered subcutaneously, intravenously, intradermally,intraperitoneally, orally, or intramuscularly. The antibody or fragmentthereof may be administered at a dose of about 0.1 mg/kg of body weightto about 100 mg/kg of body weight of the subject. In certainembodiments, an antibody of the present invention may be administered atone or more doses comprising between 50 mg to 600 mg.

The present invention also includes use of an anti-C5 antibody orantigen-binding fragment thereof of the invention in the manufacture ofa medicament for the treatment of a disease or disorder that wouldbenefit from the blockade of C5 binding and/or activity.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows inhibition of C5a levels by anti-C5 antibody H4H12166P in adose-dependent manner, as determined by ELISA (described in Example 9herein).

FIG. 2 shows total serum concentration vs. time following a single 15mg/kg intravenous injection of H4H12166P, H4H12161P, or Comparator 2 tomale cynomolgus monkeys (described in Example 10 herein).Concentration-time profiles were plotted through the first post dosebelow the limit of quantitation (BLQ) result, if applicable, which wasimputed as LLOQ/2. Each data point represents Mean (±SD) (n=4animals/group); concentrations considered to be impacted by ADA wereexcluded from 1 animal in the H4H12166P group and 1 animal in theH4H12161P group starting at Day 36 and Day 29, respectively. LLOQ=Lowerlimit of quantification

FIG. 3A and FIG. 3B show percent hemolysis vs. time in ex vivo red bloodcell (A) Classical Pathway and (B) Alternative Pathway assays followinga single intravenous injection of H4H12166P, H4H12161P or Comparator 2to male cynomolgus monkeys. % hemolysis, calculated as a ratio ofexperimental vs. maximal lysis with % background lysis subtracted fromboth values, is related to the amount of C5 inhibited by the particularanti-C5 antibody present in the serum at a given time point. Each datapoint represents Mean (±SD).

FIG. 4 shows total serum concentration vs. time profiles of selectedanti-C5 antibodies in mice humanized for C5 (described in Example 11herein). Humanized C5 mice were administered a single 15 mg/kgsubcutaneous dose of H4H12166P, Comparator 1 or Comparator 2. Each datapoint represents the mean±s.e.m. (n=4-5 each). Antibody concentrationsin sera were monitored 1, 10, 20, 30 and 40 days post injection using asandwich ELISA.

FIG. 5 shows percent hemolysis vs. time in an ex vivo complementclassical pathway hemolysis assay of selected anti-C5 antibodies in micehumanized for C5. Humanized C5 mice were administered a single 15 mg/kgsubcutaneous dose of H4H12166P, Comparator 1 or Comparator 2. Each datapoint represents the mean±s.e.m. (n=4-5 each). The percent hemolysis inserum was monitored at predose, 10, 20, 30, 40 and 50 days postinjection. % hemolysis, calculated as a ratio of experimental vs.maximal lysis with % background lysis subtracted from both values, isrelated to the amount of C5 inhibited by the particular anti-C5 antibodypresent in the serum at a given time point.

FIG. 6 shows total serum concentration vs. time profiles of selectedanti-C5 antibodies in mice humanized for C5 (described in Example 11herein). Mice were administered a single 15 mg/kg subcutaneous dose ofH4H12166P, H4H12161P, Comparator 1 or IgG4^(P) isotype control. Eachdata point represents the mean±s.e.m. (n=5 each). Antibody levels insera were monitored 6 hours, 1, 2, 3, 4, 7, 10, 14, 21, 30, 45 and 59days post injection using a sandwich ELISA.

FIG. 7 is a graph showing optical coherence tomography (OCT) scores inmice treated with isotype control or with anti-C5 antibody M1M17628N at10 mg/kg or 50 mg/kg (described in Example 14 herein). ****p<0.0001,two-way ANOVA treatment with anti-C5 antibody at 50 mg/kg vs. notreatment or treatment with isotype control.

FIG. 8 shows inhibition of classical pathway hemolysis by anti-C5antibody M1M17628N in the absence of C3 (A); and in the presence of 80μg/mL of human C3 (B) (described in Example 14 herein).

FIG. 9 shows cell cluster count in C5 humanized mice treated withisotype control or with anti-human C5 antibody H4H12170P at 10 mg/kg or50 mg/kg (described in Example 15 herein). n=8-12 eyes for each group

FIG. 10 is a graph showing OCT scores in C5 humanized mice treated withisotype control or with anti-human C5 antibody H4H12170P at 10 mg/kg or50 mg/kg (described in Example 15 herein). n=8-12 eyes for each group

FIG. 11 is a graph showing OCT scores in C5 humanized mice treated withisotype control, anti-human C5 antibody H4H12166P at 3 mg/kg or 10mg/kg, or Comparator 2 at 10 mg/kg. n=6-12 eyes for each group(described in Example 15 herein).

FIG. 12 shows cell cluster count in C5 humanized mice treated withisotype control, anti-human C5 antibody H4H12166P at 3 mg/kg or 10mg/kg, or Comparator 2 at 10 mg/kg. n=6-12 eyes for each group(described in Example 15 herein).

FIG. 13 is a survival curve of NZBWF1 mice treated with isotype controlor with anti-C5 antibodies M1M17628N or M1M17627N (described in Example17 herein).

FIG. 14A and FIG. 14B show levels of (A) urinary albumin and (B) urinaryalbumin normalized to urinary creatinine in NZBWF1 mice treated withisotype control or with anti-C5 antibodies M1M17628N or M1M17627N(described in Example 17 herein).

FIG. 15 shows levels of blood urea nitrogen in NZBWF1 mice treated withisotype control or with anti-C5 antibodies M1M17628N or M1M17627N(described in Example 17 herein).

FIG. 16 is a graph showing inhibition of antibody-dependent cytotoxicityof astrocytes by anti-C5 antibodies H4H12166P, H4H12170P, Comparator 1and Comparator 2, as described in Example 18.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

Definitions

The term “C5”, also called “complement component 5” or “complementfactor 5” refers to the serum protein of the complement cascade. The C5protein is a 1676 amino acid protein comprising two chains, alpha andbeta. The protein represents the convergence point for three complementactivation pathways: classical pathway, alternative pathway and themannose binding lectin pathway. The amino acid sequence of full-lengthC5 protein is exemplified by the amino acid sequence provided in GenBankas accession number NP_001726.2 (SEQ ID NO: 355). The term “C5” includesrecombinant C5 protein or a fragment thereof. The term also encompassesC5 protein or a fragment thereof coupled to, for example, histidine tag,mouse or human Fc, or a signal sequence such as ROR1. For example, theterm includes sequences exemplified by the sequence shown in SEQ ID NO:356 or 357, comprising a histidine tag at the C-terminal, coupled toamino acid residues 19-1676 of full-length C5 protein. The term alsoincludes protein variants that comprise a histidine tag at theC-terminal, coupled to amino acid residues 19-1676 of full-length C5protein with a R885H change or a R885C change.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds(i.e., “full antibody molecules”), as well as multimers thereof (e.g.IgM) or antigen-binding fragments thereof. Each heavy chain is comprisedof a heavy chain variable region (“HCVR” or “V_(H)”) and a heavy chainconstant region (comprised of domains C_(H)1, C_(H)2 and C_(H)3). Eachlight chain is comprised of a light chain variable region (“LCVR or“V_(L)”) and a light chain constant region (C_(L)). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, theFRs of the antibody (or antigen binding fragment thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

Substitution of one or more CDR residues or omission of one or more CDRsis also possible. Antibodies have been described in the scientificliterature in which one or two CDRs can be dispensed with for binding.Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regionsbetween antibodies and their antigens, based on published crystalstructures, and concluded that only about one fifth to one third of CDRresidues actually contact the antigen. Padlan also found many antibodiesin which one or two CDRs had no amino acids in contact with an antigen(see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

CDR residues not contacting antigen can be identified based on previousstudies (for example residues H60-H65 in CDRH2 are often not required),from regions of Kabat CDRs lying outside Chothia CDRs, by molecularmodeling and/or empirically. If a CDR or residue(s) thereof is omitted,it is usually substituted with an amino acid occupying the correspondingposition in another human antibody sequence or a consensus of suchsequences. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically. Empirical substitutions canbe conservative or non-conservative substitutions.

The fully human anti-C5 monoclonal antibodies disclosed herein maycomprise one or more amino acid substitutions, insertions and/ordeletions in the framework and/or CDR regions of the heavy and lightchain variable domains as compared to the corresponding germlinesequences. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germline sequences availablefrom, for example, public antibody sequence databases. The presentinvention includes antibodies, and antigen-binding fragments thereof,which are derived from any of the amino acid sequences disclosed herein,wherein one or more amino acids within one or more framework and/or CDRregions are mutated to the corresponding residue(s) of the germlinesequence from which the antibody was derived, or to the correspondingresidue(s) of another human germline sequence, or to a conservativeamino acid substitution of the corresponding germline residue(s) (suchsequence changes are referred to herein collectively as “germlinemutations”). A person of ordinary skill in the art, starting with theheavy and light chain variable region sequences disclosed herein, caneasily produce numerous antibodies and antigen-binding fragments whichcomprise one or more individual germline mutations or combinationsthereof. In certain embodiments, all of the framework and/or CDRresidues within the V_(H) and/or V_(L) domains are mutated back to theresidues found in the original germline sequence from which the antibodywas derived. In other embodiments, only certain residues are mutatedback to the original germline sequence, e.g., only the mutated residuesfound within the first 8 amino acids of FR1 or within the last 8 aminoacids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. In other embodiments, one or more of the framework and/or CDRresidue(s) are mutated to the corresponding residue(s) of a differentgermline sequence (i.e., a germline sequence that is different from thegermline sequence from which the antibody was originally derived).Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be easily tested for one or more desired property such as, improvedbinding specificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes fully human anti-C5 monoclonalantibodies comprising variants of any of the HCVR, LCVR, and/or CDRamino acid sequences disclosed herein having one or more conservativesubstitutions. For example, the present invention includes anti-C5antibodies having HCVR, LCVR, and/or CDR amino acid sequences with,e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservativeamino acid substitutions relative to any of the HCVR, LCVR, and/or CDRamino acid sequences disclosed herein.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human mAbs of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody”, as usedherein, is not intended to include mAbs in which CDR sequences derivedfrom the germline of another mammalian species (e.g., mouse), have beengrafted onto human FR sequences. The term includes antibodiesrecombinantly produced in a non-human mammal, or in cells of a non-humanmammal. The term is not intended to include antibodies isolated from orgenerated in a human subject.

The term “recombinant”, as used herein, refers to antibodies orantigen-binding fragments thereof of the invention created, expressed,isolated or obtained by technologies or methods known in the art asrecombinant DNA technology which include, e.g., DNA splicing andtransgenic expression. The term refers to antibodies expressed in anon-human mammal (including transgenic non-human mammals, e.g.,transgenic mice), or a cell (e.g., CHO cells) expression system orisolated from a recombinant combinatorial human antibody library.

The term “specifically binds,” or “binds specifically to”, or the like,means that an antibody or antigen-binding fragment thereof forms acomplex with an antigen that is relatively stable under physiologicconditions. Specific binding can be characterized by an equilibriumdissociation constant of at least about 1×10⁻⁸ M or less (e.g., asmaller K_(D) denotes a tighter binding). Methods for determiningwhether two molecules specifically bind are well known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,and the like. As described herein, antibodies have been identified bysurface plasmon resonance, e.g., BIACORE™, which bind specifically toC5. Moreover, multi-specific antibodies that bind to one domain in C5and one or more additional antigens or a bi-specific that binds to twodifferent regions of C5 are nonetheless considered antibodies that“specifically bind”, as used herein.

The term “high affinity” antibody refers to those mAbs having a bindingaffinity to C5, expressed as K_(D), of at least 10⁻⁸ M; preferably 10⁻⁹M; more preferably 10⁻¹⁰M, even more preferably 10⁻¹¹ M, even morepreferably 10⁻¹² M, as measured by surface plasmon resonance, e.g.,BIACORE™ or solution-affinity ELISA.

By the term “slow off rate”, “Koff” or “kd” is meant an antibody thatdissociates from C5, with a rate constant of 1×10⁻³ s⁻¹ or less,preferably 1×10⁻⁴ s⁻¹ or less, as determined by surface plasmonresonance, e.g., BIACORE™.

The terms “antigen-binding portion” of an antibody, “antigen-bindingfragment” of an antibody, and the like, as used herein, include anynaturally occurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. The terms “antigen-binding fragment” of anantibody, or “antibody fragment”, as used herein, refers to one or morefragments of an antibody that retain the ability to bind to C5 protein.

In specific embodiments, antibody or antibody fragments of the inventionmay be conjugated to a moiety such a ligand or a therapeutic moiety(“immunoconjugate”), a second anti-C5 antibody, or any other therapeuticmoiety useful for treating a C5-associated disease or disorder.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies (Abs) havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds C5, or a fragment thereof, is substantially free ofAbs that specifically bind antigens other than C5.

A “blocking antibody” or a “neutralizing antibody”, as used herein (oran “antibody that neutralizes C5 activity” or “antagonist antibody”), isintended to refer to an antibody whose binding to C5 results ininhibition of at least one biological activity of C5. For example, anantibody of the invention may prevent or block complement-mediatedhemolysis by classical pathway or alternative pathway.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timebiomolecular interactions by detection of alterations in proteinconcentrations within a biosensor matrix, for example using the BIACORE™system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. The term“epitope” also refers to a site on an antigen to which B and/or T cellsrespond. It also refers to a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

The term “cross-competes”, as used herein, means an antibody orantigen-binding fragment thereof binds to an antigen and inhibits orblocks the binding of another antibody or antigen-binding fragmentthereof. The term also includes competition between two antibodies inboth orientations, i.e., a first antibody that binds and blocks bindingof second antibody and vice-versa. In certain embodiments, the firstantibody and second antibody may bind to the same epitope.Alternatively, the first and second antibodies may bind to different,but overlapping epitopes such that binding of one inhibits or blocks thebinding of the second antibody, e.g., via steric hindrance.Cross-competition between antibodies may be measured by methods known inthe art, for example, by a real-time, label-free bio-layerinterferometry assay. Cross-competition between two antibodies may beexpressed as the binding of the second antibody that is less than thebackground signal due to self-self binding (wherein first and secondantibodies is the same antibody). Cross-competition between 2 antibodiesmay be expressed, for example, as % binding of the second antibody thatis less than the baseline self-self background binding (wherein firstand second antibodies is the same antibody).

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or GAP, as discussed below. A nucleic acid molecule havingsubstantial identity to a reference nucleic acid molecule may, incertain instances, encode a polypeptide having the same or substantiallysimilar amino acid sequence as the polypeptide encoded by the referencenucleic acid molecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 90% sequence identity, even more preferably atleast 95%, 98% or 99% sequence identity. Preferably, residue positions,which are not identical, differ by conservative amino acidsubstitutions. A “conservative amino acid substitution” is one in whichan amino acid residue is substituted by another amino acid residuehaving a side chain (R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment are wellknown to those of skill in the art. See, e.g., Pearson (1994) MethodsMol. Biol. 24: 307-331, which is herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartate and glutamate, and 7) sulfur-containingside chains: cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443 45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA with default or recommended parameters; a program in GCG Version6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson (2000) supra). Another preferred algorithmwhen comparing a sequence of the invention to a database containing alarge number of sequences from different organisms is the computerprogram BLAST, especially BLASTP or TBLASTN, using default parameters.See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997)Nucleic Acids Res. 25:3389-3402, each of which is herein incorporated byreference.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

As used herein, the term “subject” refers to an animal, preferably amammal, more preferably a human, in need of amelioration, preventionand/or treatment of a C5-associated disease or disorder such as atypicalhemolytic uremic syndrome (aHUS) or paroxysmal nocturnal hemoglobinuria(PNH). The term includes human subjects who have or are at risk ofhaving such a disease or disorder.

As used herein, the terms “treat”, “treating”, or “treatment” refer tothe reduction or amelioration of the severity of at least one symptom orindication of a C5-associated disease or disorder due to theadministration of a therapeutic agent such as an antibody of the presentinvention to a subject in need thereof. The terms include inhibition ofprogression of disease or of worsening of a symptom/indication. Theterms also include positive prognosis of disease, i.e., the subject maybe free of disease or may have reduced disease upon administration of atherapeutic agent such as an antibody of the present invention. Thetherapeutic agent may be administered at a therapeutic dose to thesubject.

The terms “prevent”, “preventing” or “prevention” refer to inhibition ofmanifestation of a C5-associated disease or disorder or any symptoms orindications of such a disease or disorder upon administration of anantibody of the present invention.

Antigen-Binding Fragments of Antibodies

Unless specifically indicated otherwise, the term “antibody,” as usedherein, shall be understood to encompass antibody molecules comprisingtwo immunoglobulin heavy chains and two immunoglobulin light chains(i.e., “full antibody molecules”) as well as antigen-binding fragmentsthereof. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, as used herein,include any naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein that specificallybinds an antigen to form a complex. The terms “antigen-binding fragment”of an antibody, or “antibody fragment”, as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to C5 protein. An antibody fragment may include a Fab fragment, aF(ab′)₂ fragment, a Fv fragment, a dAb fragment, a fragment containing aCDR, or an isolated CDR. In certain embodiments, the term“antigen-binding fragment” refers to a polypeptide fragment of amulti-specific antigen-binding molecule. Antigen-binding fragments of anantibody may be derived, e.g., from full antibody molecules using anysuitable standard techniques such as proteolytic digestion orrecombinant genetic engineering techniques involving the manipulationand expression of DNA encoding antibody variable and (optionally)constant domains. Such DNA is known and/or is readily available from,e.g., commercial sources, DNA libraries (including, e.g., phage-antibodylibraries), or can be synthesized. The DNA may be sequenced andmanipulated chemically or by using molecular biology techniques, forexample, to arrange one or more variable and/or constant domains into asuitable configuration, or to introduce codons, create cysteineresidues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDR,which is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)—C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemono-specific or multi-specific (e.g., bi-specific). A multi-specificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multi-specific antibody format, including theexemplary bi-specific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind to C5protein.

An immunogen comprising any one of the following can be used to generateantibodies to C5 protein. In certain embodiments, the antibodies of theinvention are obtained from mice immunized with a full length, native C5protein (See, for example, GenBank accession number NP_001726.2) (SEQ IDNO: 355), or with DNA encoding the protein or fragment thereof.Alternatively, the protein or a fragment thereof may be produced usingstandard biochemical techniques and modified and used as immunogen. Incertain embodiments of the invention, the immunogen is a fragment of C5protein that ranges from about amino acid residues 19-1676 of SEQ ID NO:355.

In some embodiments, the immunogen may be a recombinant C5 protein orfragment thereof expressed in E. coli or in any other eukaryotic ormammalian cells such as Chinese hamster ovary (CHO) cells.

Using VELOCIMMUNE® technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other knownmethod for generating monoclonal antibodies, high affinity chimericantibodies to C5 are initially isolated having a human variable regionand a mouse constant region. The VELOCIMMUNE® technology involvesgeneration of a transgenic mouse having a genome comprising human heavyand light chain variable regions operably linked to endogenous mouseconstant region loci such that the mouse produces an antibody comprisinga human variable region and a mouse constant region in response toantigenic stimulation. The DNA encoding the variable regions of theheavy and light chains of the antibody are isolated and operably linkedto DNA encoding the human heavy and light chain constant regions. TheDNA is then expressed in a cell capable of expressing the fully humanantibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. As in the experimentalsection below, the antibodies are characterized and selected fordesirable characteristics, including affinity, selectivity, epitope,etc. The mouse constant regions are replaced with a desired humanconstant region to generate the fully human antibody of the invention,for example wild-type or modified IgG1 or IgG4. While the constantregion selected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region.

Bioequivalents

The anti-C5 antibodies and antibody fragments of the present inventionencompass proteins having amino acid sequences that vary from those ofthe described antibodies, but that retain the ability to bind C5protein. Such variant antibodies and antibody fragments comprise one ormore additions, deletions, or substitutions of amino acids when comparedto parent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described antibodies. Likewise, theantibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an antibody or antibody fragment that is essentiallybioequivalent to an antibody or antibody fragment of the invention.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single dose or multipledoses. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,or potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and/or in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of the antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include antibody variants comprising aminoacid changes, which modify the glycosylation characteristics of theantibodies, e.g., mutations that eliminate or remove glycosylation.

Anti-C5 Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-C5antibodies are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes anti-C5 antibodies comprising a mutation inthe C_(H)2 or a C_(H)3 region of the Fc domain, wherein the mutation(s)increases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Suchmutations may result in an increase in serum half-life of the antibodywhen administered to an animal. Non-limiting examples of such Fcmodifications include, e.g., a modification at position 250 (e.g., E orQ); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., Sor T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., A, W, H, F or Y[N434A, N434W, N434H, N434F or N434Y]); or a modification at position250 and/or 428; or a modification at position 307 or 308 (e.g., 308F,V308F), and 434. In one embodiment, the modification comprises a 428L(e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g.,V2591), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T,and 256E) modification; a 250Q and 428L modification (e.g., T250Q andM428L); and a 307 and/or 308 modification (e.g., 308F or 308P). In yetanother embodiment, the modification comprises a 265A (e.g., D265A)and/or a 297A (e.g., N297A) modification.

For example, the present invention includes anti-C5 antibodiescomprising an Fc domain comprising one or more pairs or groups ofmutations selected from the group consisting of: 250Q and 248L (e.g.,T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);428L and 434S (e.g., M428L and N434S); 2571 and 3111 (e.g., P2571 andQ3111); 2571 and 434H (e.g., P2571 and N434H); 376V and 434H (e.g.,D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A);and 433K and 434F (e.g., H433K and N434F). All possible combinations ofthe foregoing Fc domain mutations and other mutations within theantibody variable domains disclosed herein, are contemplated within thescope of the present invention.

The present invention also includes anti-C5 antibodies comprising achimeric heavy chain constant (C_(H)) region, wherein the chimeric C_(H)region comprises segments derived from the C_(H) regions of more thanone immunoglobulin isotype. For example, the antibodies of the inventionmay comprise a chimeric C_(H) region comprising part or all of a C_(H)2domain derived from a human IgG1, human IgG2 or human IgG4 molecule,combined with part or all of a C_(H)3 domain derived from a human IgG1,human IgG2 or human IgG4 molecule. According to certain embodiments, theantibodies of the invention comprise a chimeric C_(H) region having achimeric hinge region. For example, a chimeric hinge may comprise an“upper hinge” amino acid sequence (amino acid residues from positions216 to 227 according to EU numbering) derived from a human IgG1, a humanIgG2 or a human IgG4 hinge region, combined with a “lower hinge”sequence (amino acid residues from positions 228 to 236 according to EUnumbering) derived from a human IgG1, a human IgG2 or a human IgG4 hingeregion. According to certain embodiments, the chimeric hinge regioncomprises amino acid residues derived from a human IgG1 or a human IgG4upper hinge and amino acid residues derived from a human IgG2 lowerhinge. An antibody comprising a chimeric C_(H) region as describedherein may, in certain embodiments, exhibit modified Fc effectorfunctions without adversely affecting the therapeutic or pharmacokineticproperties of the antibody. (See, e.g., U.S. Patent ApplicationPublication 2014/0243504, the disclosure of which is hereby incorporatedby reference in its entirety).

Biological Characteristics of the Antibodies

In general, the antibodies of the present invention function by bindingto C5 protein and preventing its cleavage to C5a and C5b. For example,the present invention includes antibodies and antigen-binding fragmentsof antibodies that bind C5 protein (e.g., at 25° C. or at 37° C.) with aK_(D) of less than 9 nM as measured by surface plasmon resonance, e.g.,using the assay format as defined in Example 3 herein. In certainembodiments, the antibodies or antigen-binding fragments thereof bind C5with a K_(D) of less than about 9 nM, less than about 5 nM, less thanabout 2 nM, less than about 1 nM, less than about 500 pM, less than 250pM, or less than 100 pM, as measured by surface plasmon resonance, e.g.,using the assay format as defined in Example 3 herein, or asubstantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind human C5 protein with a dissociativehalf-life (t½) of greater than about 2 minutes as measured by surfaceplasmon resonance at 25° C., e.g., using an assay format as defined inExample 4 herein, or a substantially similar assay. In certainembodiments, the antibodies or antigen-binding fragments of the presentinvention bind C5 protein with a t½ of greater than about 5 minutes,greater than about 10 minutes, greater than about 30 minutes, greaterthan about 50 minutes, greater than about 100 minutes, greater thanabout 150 minutes, greater than about 200 minutes, or greater than about250 minutes, as measured by surface plasmon resonance at 25° C., e.g.,using an assay format as defined in Example 3 herein (e.g., mAb-captureor antigen-capture format), or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind human C5 protein with a dissociativehalf-life (t½) of greater than about 1.5 minutes as measured by surfaceplasmon resonance at 37° C., e.g., using an assay format as defined inExample 4 herein, or a substantially similar assay. In certainembodiments, the antibodies or antigen-binding fragments of the presentinvention bind C5 protein with a t½ of greater than about 2 minutes,greater than about 5 minutes, greater than about 10 minutes, greaterthan about 25 minutes, greater than about 50 minutes, greater than about100 minutes, greater than about 150 minutes, or greater than about 200minutes, as measured by surface plasmon resonance at 37° C., e.g., usingan assay format as defined in Example 3 herein (e.g., mAb-capture orantigen-capture format), or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that bind monkey C5 protein (e.g., at 25° C. orat 37° C.) with a K_(D) of less than 120 nM as measured by surfaceplasmon resonance, e.g., using the assay format as defined in Example 3herein. In certain embodiments, the antibodies or antigen-bindingfragments thereof bind monkey C5 with a K_(D) of less than about 120 nM,less than about 100 nM, less than about 50 nM, less than about 25 nM,less than about 10 nM, less than about 5 nM, less than about 1 nM, lessthan about 500 pM, or less than 250 pM, as measured by surface plasmonresonance, e.g., using the assay format as defined in Example 3 herein,or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that bind modified human C5 protein with R885Hchange (exemplified by SEQ ID NO: 356) with a K_(D) of less than 70 nMas measured by surface plasmon resonance, e.g., using the assay formatas defined in Example 3 herein. C5 variants have shown poor response toanti-C5 antibodies previously disclosed in the art (e.g., Nishimura etal 2014, New Engl. J. Med. 370: 632-639). In certain embodiments, theantibodies or antigen-binding fragments thereof bind the modified humanC5 with a K_(D) of less than about 65 nM, less than about 50 nM, lessthan about 20 nM, less than about 10 nM, less than about 5 nM, less thanabout 3 nM, or less than 2 nM, as measured by surface plasmon resonance,e.g., using the assay format as defined in Example 3 herein, or asubstantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that bind modified human C5 protein with R885Cchange (exemplified by SEQ ID NO: 357) with a K_(D) of less than 160 nMas measured by surface plasmon resonance, e.g., using the assay formatas defined in Example 3 herein. C5 variants have shown poor response toanti-C5 antibodies previously disclosed in the art (e.g., Nishimura etal 2014, New Engl. J. Med. 370: 632-639). In certain embodiments, theantibodies or antigen-binding fragments thereof bind the modified humanC5 with a K_(D) of less than about 150 nM, less than about 100 nM, lessthan about 50 nM, less than about 20 nM, less than about 10 nM, lessthan about 5 nM, or less than 2 nM, as measured by surface plasmonresonance, e.g., using the assay format as defined in Example 3 herein,or a substantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments of antibodies that inhibit complement dependent cytotoxicity(CDC) with IC₅₀ less than 10 nM as measured by a luminescence assay,e.g., using the assay format as defined in Example 6 herein. In certainembodiments, the antibodies or antigen-binding fragments thereof inhibitCDC with IC₅₀ less than about 5 nM, less than about 3.5 nM, or less thanabout 2 nM, as measured by a B-cell luminescence assay, e.g., using theassay format as defined in Example 6 herein, or a substantially similarassay.

The present invention also includes antibodies and antigen-bindingfragments that block human C5-mediated classical pathway (CP) hemolysisby more than 94% and with an IC₅₀ less than 6 nM, as measured by a CPhemolysis assay, e.g., using the assay format as defined in Example 8herein. In certain embodiments, the antibodies or antigen-bindingfragments thereof block CP hemolysis with IC₅₀ less than about 6 nM,less than about 5 nM, less than about 4 nM, less than about 3 nM, orless than about 2 nM, as measured by CP hemolysis assay, e.g., using theassay format as defined in Example 8 herein, or a substantially similarassay.

The present invention also includes antibodies and antigen-bindingfragments that block human C5-mediated alternative pathway (AP)hemolysis by more than 70% and with an IC₅₀ less than 165 nM, asmeasured by a AP hemolysis assay, e.g., using the assay format asdefined in Example 8 herein. In certain embodiments, the antibodies orantigen-binding fragments thereof block AP hemolysis with IC₅₀ less thanabout 160 nM, less than about 150 nM, less than about 100 nM, less thanabout 50 nM, or less than about 20 nM, as measured by AP hemolysisassay, e.g., using the assay format as defined in Example 8 herein, or asubstantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments that block African green monkey C5-mediated classical pathway(CP) hemolysis by more than 40% and with an IC₅₀ less than 185 nM, asmeasured by a CP hemolysis assay, e.g., using the assay format asdefined in Example 8 herein. In certain embodiments, the antibodies orantigen-binding fragments thereof block CP hemolysis with IC₅₀ less thanabout 180 nM, less than about 150 nM, less than about 100 nM, less thanabout 75 nM, or less than about 50 nM, as measured by CP hemolysisassay, e.g., using the assay format as defined in Example 8 herein, or asubstantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments that block African green monkey C5-mediated alternativepathway (AP) hemolysis with an IC₅₀ less than 235 nM, as measured by aAP hemolysis assay, e.g., using the assay format as defined in Example 8herein. In certain embodiments, the antibodies or antigen-bindingfragments thereof block AP hemolysis with IC₅₀ less than about 200 nM,less than about 150 nM, less than about 100 nM, less than about 50 nM,or less than about 20 nM, as measured by AP hemolysis assay, e.g., usingthe assay format as defined in Example 8 herein, or a substantiallysimilar assay.

The present invention also includes antibodies and antigen-bindingfragments that block more than 90% of cynomolgus monkey C5-mediatedclassical pathway (CP) hemolysis with an IC₅₀ less than 145 nM, asmeasured by a CP hemolysis assay, e.g., using the assay format asdefined in Example 8 herein. In certain embodiments, the antibodies orantigen-binding fragments thereof block CP hemolysis with IC₅₀ less thanabout 140 nM, less than about 120 nM, less than about 100 nM, less thanabout 75 nM, or less than about 50 nM, as measured by CP hemolysisassay, e.g., using the assay format as defined in Example 8 herein, or asubstantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments that block cynomolgus monkey C5-mediated alternative pathway(AP) hemolysis with an IC₅₀ less than 30 nM, as measured by an APhemolysis assay, e.g., using the assay format as defined in Example 8herein. In certain embodiments, the antibodies or antigen-bindingfragments thereof block AP hemolysis with IC₅₀ less than about 25 nM,less than about 20 nM, less than about 10 nM, less than about 5 nM, orless than about 2 nM, as measured by AP hemolysis assay, e.g., using theassay format as defined in Example 8 herein, or a substantially similarassay.

The present invention also includes antibodies and antigen-bindingfragments that show improved pharmacokinetic (PK) and pharmacodynamic(PD) properties as compared to anti-C5 antibodies in the art. Theanti-C5 antibodies of the present invention show less susceptibility totarget-mediated clearance upon administration, as shown in Examples 9and 10 herein. In certain embodiments, the present invention includesanti-C5 antibodies and antigen-binding fragments thereof that show serumconcentrations for extended periods, e.g., more than 20 days, more than25 days, more than 30 days, more than 35 days, more than 40 days, morethan 45 days, more than 50 days, more than 55 days, or more than 60days, as described in Examples 9 and 10 herein. In certain embodiments,the anti-C5 antibodies of the present invention show an extended serumhalf-life of more than 10 days, as compared to anti-C5 antibodies in theart.

In certain embodiments, the present invention provides anti-C5antibodies and antigen-binding fragments thereof that have high affinityfor human C5 (e.g., K_(D) less than 0.3 nM) and a lower clearance (e.g.,extended serum half-life, improved pharmacodynamic activity over moredays than previously known anti-C5 antibodies). Such antibodies of thepresent invention may be advantageously used with less frequent dosingin a subject with a C5-associated disease or disorder.

In one embodiment, the present invention provides an isolatedrecombinant antibody or antigen-binding fragment thereof that bindsspecifically to C5 protein, wherein the antibody or fragment thereofexhibits one or more of the following characteristics: (a) is a fullyhuman monoclonal antibody; (b) binds to human C5 with a dissociationconstant (K_(D)) of less than 0.9 nM at 25° C., as measured in a surfaceplasmon resonance assay; (c) binds to human C5 with a K_(D) of less than0.3 nM at 37° C., as measured in a surface plasmon resonance assay; (d)has serum concentration of more than 10 μg/mL through day 70 uponadministration to cynomolgus monkey; (e) blocks CP- and AP hemolysisthrough day 35 upon administration to cynomolgus monkey, as measured inan ex vivo hemolysis assay; (f) has serum half-life of more than 10 daysin cynomolgus monkey; (g) has serum concentration of more than 10 μg/mLthrough day 40 upon administration to C5-humanized mice; (h) blocks CPhemolysis through day 30 upon administration to C5-humanized mice, asmeasured in an ex vivo hemolysis assay; and (i) has serum half-life ofmore than 10 days in C5-humanized mice.

In one embodiment, the present invention provides an isolatedrecombinant antibody or antigen-binding fragment thereof that bindsspecifically to C5 protein, wherein the antibody or fragment thereofexhibits one or more of the following characteristics: (a) is a fullyhuman monoclonal antibody; (b) binds to human C5 with a dissociationconstant (K_(D)) of less than 0.9 nM at 25° C., as measured in a surfaceplasmon resonance assay; (c) binds to human C5 with a K_(D) of less than0.3 nM at 37° C., as measured in a surface plasmon resonance assay; (d)binds to monkey C5 with a K_(D) of less than 65 nM, as measured in asurface plasmon resonance assay; (e) binds to human C5 variant R885H(SEQ ID NO: 356) with a K_(D) of less than 0.5 nM, as measured in asurface plasmon resonance assay; (f) binds to human C5 variant R885C(SEQ ID NO: 357) with a K_(D) of less than 0.5 nM, as measured in asurface plasmon resonance assay; (g) blocks human C5-mediated classicalpathway (CP) hemolysis by more than 95% and with IC₅₀ less than 6 nM, asmeasured in a CP hemolysis assay; (h) blocks human C5-mediatedalternative pathway (AP) hemolysis by more than 70% and with IC₅₀ lessthan 165 nM, as measured in a AP hemolysis assay; (i) inhibits Africangreen monkey C5-mediated CP hemolysis with IC₅₀ less than 185 nM, asmeasured in a CP hemolysis assay; (j) inhibits African green monkeyC5-mediated AP hemolysis with IC₅₀ less than 235 nM, as measured in a APhemolysis assay; (k) inhibits cynomolgus monkey C5-mediated CP hemolysiswith IC₅₀ less than 145 nM, as measured in a CP hemolysis assay; and (I)inhibits cynomolgus monkey C5-mediated AP hemolysis with IC₅₀ less than30 nM, as measured in a AP hemolysis assay.

The antibodies of the present invention may possess one or more of theaforementioned biological characteristics, or any combinations thereof.Other biological characteristics of the antibodies of the presentinvention will be evident to a person of ordinary skill in the art froma review of the present disclosure including the working Examplesherein.

Epitope Mapping and Related Technologies

The present invention includes anti-C5 antibodies which interact withone or more amino acids found within one or more regions of the C5protein molecule including, the alpha polypeptide and beta polypeptide.The epitope to which the antibodies bind may consist of a singlecontiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within anyof the aforementioned domains of the C5 protein molecule (e.g. a linearepitope in a domain). Alternatively, the epitope may consist of aplurality of non-contiguous amino acids (or amino acid sequences)located within either or both of the aforementioned domains of theprotein molecule (e.g. a conformational epitope).

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,for example, routine cross-blocking assays, such as that described inAntibodies, Harlow and Lane (Cold Spring Harbor Press, Cold SpringHarbor, N.Y.). Other methods include alanine scanning mutationalanalysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis crystallographic studies and NMRanalysis. In addition, methods such as epitope excision, epitopeextraction and chemical modification of antigens can be employed (Tomer(2000) Prot. Sci. 9: 487-496). Another method that can be used toidentify the amino acids within a polypeptide with which an antibodyinteracts is hydrogen/deuterium exchange detected by mass spectrometry.In general terms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water and exchangeable protons within aminoacids that are protected by the antibody complex undergodeuterium-to-hydrogen back-exchange at a slower rate than exchangeableprotons within amino acids that are not part of the interface. As aresult, amino acids that form part of the protein/antibody interface mayretain deuterium and therefore exhibit relatively higher mass comparedto amino acids not included in the interface. After dissociation of theantibody, the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antibodies (mAbs) directed against the sameantigen according to the similarities of the binding profile of eachantibody to chemically or enzymatically modified antigen surfaces (seeUS 2004/0101920, herein specifically incorporated by reference in itsentirety). Each category may reflect a unique epitope either distinctlydifferent from or partially overlapping with epitope represented byanother category. This technology allows rapid filtering of geneticallyidentical antibodies, such that characterization can be focused ongenetically distinct antibodies. When applied to hybridoma screening,MAP may facilitate identification of rare hybridoma clones that producemAbs having the desired characteristics. MAP may be used to sort theantibodies of the invention into groups of antibodies binding differentepitopes.

In certain embodiments, the anti-C5 antibodies or antigen-bindingfragments thereof bind an epitope within any one or more of the regionsexemplified in C5 protein, either in natural form, as exemplified in SEQID NO: 355, or recombinantly produced, or to a fragment thereof. In someembodiments, the antibodies of the invention bind to a region comprisingone or more amino acids selected from the group consisting of amino acidresidues 19-1676 of human C5 protein.

In certain embodiments, the antibodies of the invention, interact withat least one amino acid sequence selected from the group consisting ofamino acid residues ranging from about position 19 to about position750; or amino acid residues ranging from about position 751 to aboutposition 1676 of SEQ ID NO: 355.

In certain embodiments, the present invention includes anti-C5antibodies and antigen-binding fragments thereof that interact with oneor more epitopes found within the alpha and/or the beta chain of C5 (SEQID NO: 359). The epitope(s) may consist of one or more contiguoussequences of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more) amino acids located within alpha chainand/or beta chain of C5. Alternatively, the epitope may consist of aplurality of non-contiguous amino acids (or amino acid sequences)located within C5. As shown in Example 11 herein, the epitope of C5 withwhich the exemplary antibody of the invention H4H12166P interacts isdefined by: (i) the amino acid sequence NMATGMDSW (SEQ ID NO: 360) whichcorresponds to amino acids 591 to 599 comprised in the beta chain of SEQID NO: 359; and (ii) the amino acid sequence WEVHLVPRRKQLQFALPDSL (SEQID NO: 361), which corresponds to amino acids 775 to 794 comprised inthe alpha chain of SEQ ID NO: 359. Accordingly, the present inventionincludes anti-C5 antibodies that interact with one or more amino acidscontained within the region consisting of (i) the amino acid sequenceNMATGMDSW (SEQ ID NO: 360), which corresponds to amino acids 591 to 599of SEQ ID NO: 359; and (ii) the amino acid sequence WEVHLVPRRKQLQFALPDSL(SEQ ID NO: 361), which corresponds to amino acids 775 to 794 of SEQ IDNO: 359.

The present invention includes anti-C5 antibodies that bind to the sameepitope, or a portion of the epitope, as any of the specific exemplaryantibodies listed in Table 1. Likewise, the present invention alsoincludes anti-C5 antibodies that compete for binding to C5 protein or afragment thereof with any of the specific exemplary antibodies listed inTable 1. For example, the present invention includes anti-C5 antibodiesthat cross-compete for binding to C5 protein with one or more antibodieslisted in Table 1.

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-C5 antibody by usingroutine methods known in the art. For example, to determine if a testantibody binds to the same epitope as a reference anti-C5 antibody ofthe invention, the reference antibody is allowed to bind to a C5 proteinor peptide under saturating conditions. Next, the ability of a testantibody to bind to the C5 protein molecule is assessed. If the testantibody is able to bind to C5 following saturation binding with thereference anti-C5 antibody, it can be concluded that the test antibodybinds to a different epitope than the reference anti-C5 antibody. On theother hand, if the test antibody is not able to bind to the C5 proteinfollowing saturation binding with the reference anti-C5 antibody, thenthe test antibody may bind to the same epitope as the epitope bound bythe reference anti-C5 antibody of the invention.

To determine if an antibody competes for binding with a referenceanti-C5 antibody, the above-described binding methodology is performedin two orientations: In a first orientation, the reference antibody isallowed to bind to a C5 protein under saturating conditions followed byassessment of binding of the test antibody to the C5 molecule. In asecond orientation, the test antibody is allowed to bind to a C5molecule under saturating conditions followed by assessment of bindingof the reference antibody to the C5 molecule. If, in both orientations,only the first (saturating) antibody is capable of binding to the C5molecule, then it is concluded that the test antibody and the referenceantibody compete for binding to C5. As will be appreciated by a personof ordinary skill in the art, an antibody that competes for binding witha reference antibody may not necessarily bind to the identical epitopeas the reference antibody, but may sterically block binding of thereference antibody by binding an overlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50% but preferably 75%, 90% or even 99%as measured in a competitive binding assay (see, e.g., Junghans et al.,Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have thesame epitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other. Two antibodies have overlapping epitopes if some aminoacid mutations that reduce or eliminate binding of one antibody reduceor eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art.

Immunoconjugates

The invention encompasses a human anti-C5 monoclonal antibody conjugatedto a therapeutic moiety (“immunoconjugate”), to treat a C5-associateddisease or disorder (e.g., atypical hemolytic uremic syndrome). As usedherein, the term “immunoconjugate” refers to an antibody which ischemically or biologically linked to a radioactive agent, a cytokine, aninterferon, a target or reporter moiety, an enzyme, a peptide or proteinor a therapeutic agent. The antibody may be linked to the radioactiveagent, cytokine, interferon, target or reporter moiety, enzyme, peptideor therapeutic agent at any location along the molecule so long as it isable to bind its target. Examples of immunoconjugates include antibodydrug conjugates and antibody-toxin fusion proteins. In one embodiment,the agent may be a second different antibody to C5 protein. The type oftherapeutic moiety that may be conjugated to the anti-C5 antibody andwill take into account the condition to be treated and the desiredtherapeutic effect to be achieved. Examples of suitable agents forforming immunoconjugates are known in the art; see for example, WO05/103081.

Multi-specific Antibodies

The antibodies of the present invention may be mono-specific,bi-specific, or multi-specific. Multi-specific antibodies may bespecific for different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244.

Any of the multi-specific antigen-binding molecules of the invention, orvariants thereof, may be constructed using standard molecular biologicaltechniques (e.g., recombinant DNA and protein expression technology), aswill be known to a person of ordinary skill in the art.

In some embodiments, C5-specific antibodies are generated in abi-specific format (a “bi-specific”) in which variable regions bindingto distinct domains of C5 protein are linked together to conferdual-domain specificity within a single binding molecule. Appropriatelydesigned bi-specifics may enhance overall C5-protein inhibitory efficacythrough increasing both specificity and binding avidity. Variableregions with specificity for individual domains, (e.g., segments of theN-terminal domain), or that can bind to different regions within onedomain, are paired on a structural scaffold that allows each region tobind simultaneously to the separate epitopes, or to different regionswithin one domain. In one example for a bi-specific, heavy chainvariable regions (V_(H)) from a binder with specificity for one domainare recombined with light chain variable regions (V_(L)) from a seriesof binders with specificity for a second domain to identify non-cognateV_(L) partners that can be paired with an original V_(H) withoutdisrupting the original specificity for that V_(H). In this way, asingle V_(L) segment (e.g., V_(L)1) can be combined with two differentV_(H) domains (e.g., V_(H)1 and V_(H)2) to generate a bi-specificcomprised of two binding “arms” (V_(H)1-V_(L)1 and V_(H)2-V_(L)1). Useof a single V_(L) segment reduces the complexity of the system andthereby simplifies and increases efficiency in cloning, expression, andpurification processes used to generate the bi-specific (See, forexample, U.S. Ser. No. 13/022,759 and US2010/0331527).

Alternatively, antibodies that bind more than one domains and a secondtarget, such as, but not limited to, for example, a second differentanti-C5 antibody, may be prepared in a bi-specific format usingtechniques described herein, or other techniques known to those skilledin the art. Antibody variable regions binding to distinct regions may belinked together with variable regions that bind to relevant sites on,for example, the extracellular domain of C5, to confer dual-antigenspecificity within a single binding molecule. Appropriately designedbi-specifics of this nature serve a dual function. Variable regions withspecificity for the extracellular domain are combined with a variableregion with specificity for outside the extracellular domain and arepaired on a structural scaffold that allows each variable region to bindto the separate antigens.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bi-specific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E,L358M, N384S, K392N, V397M, and V4221 by EU) in the case of IgG1antibodies; N44S, K52N, and V821 (IMGT; N384S, K392N, and V4221 by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V821 (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V4221by EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Other exemplary bispecific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab,(SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab(DAF)-IgG, and Mab² bispecific formats (see, e.g., Klein et al. 2012,mAbs 4:6, 1-11, and references cited therein, for a review of theforegoing formats). Bispecific antibodies can also be constructed usingpeptide/nucleic acid conjugation, e.g., wherein unnatural amino acidswith orthogonal chemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising the anti-C5antibodies or antigen-binding fragments thereof of the presentinvention. Therapeutic compositions in accordance with the inventionwill be administered with suitable carriers, excipients, and otheragents that are incorporated into formulations to provide improvedtransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. See also Powell etal. “Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose of antibody may vary depending upon the age and the size of asubject to be administered, target disease, conditions, route ofadministration, and the like. When an antibody of the present inventionis used for treating a disease or disorder in an adult patient, or forpreventing such a disease, it is advantageous to administer the antibodyof the present invention normally at a single dose of about 0.1 to about100 mg/kg body weight, more preferably about 5 to about 80, about 10 toabout 70, or about 20 to about 50 mg/kg body weight. Depending on theseverity of the condition, the frequency and the duration of thetreatment can be adjusted. In certain embodiments, the antibody orantigen-binding fragment thereof of the invention can be administered asan initial dose of at least about 0.1 mg to about 800 mg, about 1 toabout 600 mg, about 5 to about 500 mg, or about 10 to about 400 mg. Incertain embodiments, the initial dose may be followed by administrationof a second or a plurality of subsequent doses of the antibody orantigen-binding fragment thereof in an amount that can be approximatelythe same or less than that of the initial dose, wherein the subsequentdoses are separated by at least 1 day to 3 days; at least one week, atleast 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; atleast 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; atleast 10 weeks; at least 12 weeks; or at least 14 weeks.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, transdermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. The pharmaceutical composition can be also deliveredin a vesicle, in particular a liposome (see, for example, Langer (1990)Science 249:1527-1533).

The use of nanoparticles to deliver the antibodies of the presentinvention is also contemplated herein. Antibody-conjugated nanoparticlesmay be used both for therapeutic and diagnostic applications.Antibody-conjugated nanoparticles and methods of preparation and use aredescribed in detail by Arruebo, M., et al. 2009 (“Antibody-conjugatednanoparticles for biomedical applications” in J. Nanomat. Volume 2009,Article ID 439389, 24 pages, doi: 10.1155/2009/439389), incorporatedherein by reference. Nanoparticles may be developed and conjugated toantibodies contained in pharmaceutical compositions to target cells.Nanoparticles for drug delivery have also been described in, forexample, U.S. Pat. No. 8,257,740, or U.S. Pat. No. 8,246,995, eachincorporated herein in its entirety.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used.In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe composition's target, thus requiring only a fraction of the systemicdose.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous, intracranial, intraperitoneal andintramuscular injections, drip infusions, etc. These injectablepreparations may be prepared by methods publicly known. For example, theinjectable preparations may be prepared, e.g., by dissolving, suspendingor emulsifying the antibody or its salt described above in a sterileaqueous medium or an oily medium conventionally used for injections. Asthe aqueous medium for injections, there are, for example, physiologicalsaline, an isotonic solution containing glucose and other auxiliaryagents, etc., which may be used in combination with an appropriatesolubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol(e.g., propylene glycol, polyethylene glycol), a nonionic surfactant[e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct ofhydrogenated castor oil)], etc. As the oily medium, there are employed,e.g., sesame oil, soybean oil, etc., which may be used in combinationwith a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.The injection thus prepared is preferably filled in an appropriateampoule.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET™ (Haselmeier,Stuttgart, Germany), the EPIPEN (Dey, L.P.) and the HUMIRA™ Pen (AbbottLabs, Abbott Park, Ill.), to name only a few.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the antibody contained isgenerally about 5 to about 500 mg per dosage form in a unit dose;especially in the form of injection, it is preferred that the antibodyis contained in about 5 to about 300 mg and in about 10 to about 300 mgfor the other dosage forms.

Therapeutic Uses of the Antibodies

The antibodies of the present invention are useful for the treatment,and/or prevention of a disease or disorder or condition associated withC5 and/or for ameliorating at least one symptom associated with suchdisease, disorder or condition. In certain embodiments, an antibody orantigen-binding fragment thereof of the invention may be administered ata therapeutic dose to a patient with a disease or disorder or conditionassociated with C5.

In certain embodiments, the antibodies of the present invention areuseful in treating or preventing a symptom or indication of atypicalhemolytic uremic syndrome (aHUS). Symptoms and indications of aHUSinclude, but are not limited to, platelet activation, hemolysis,systemic thrombotic microangiopathy (formation of blood clots in smallblood vessels throughout the body) leading to stroke, heart attack,kidney failure and/or death, end-stage renal disease, permanent renaldamage, abdominal pain, confusion, edema, fatigue, nausea/vomiting,diarrhea, and microangiopathic anemia.

In certain embodiments, the antibodies of the present invention areuseful in treating or preventing a symptom or indication of paroxysmalnocturnal hemoglobinuria (PNH). Symptoms and indications of PNH include,but are not limited to, destruction of red blood cells, thrombosis(including deep vein thrombosis, pulmonary embolism), intravascularhemolytic anemia, red discoloration of urine, symptoms of anemia such astiredness, shortness of breath, and palpitations, abdominal pain anddifficulty swallowing.

In certain embodiments, the antibodies of the present invention areuseful for treating or preventing at least one symptom or indication ofa C5-associated disease or disorder selected from the group consistingof neurological disorders, renal disorders, multiple sclerosis, stroke,Guillain Barre Syndrome, traumatic brain injury, Parkinson's disease,disorders of inappropriate or undesirable complement activation,hemodialysis complications, hyperacute allograft rejection, xenograftrejection, interleukin-2 induced toxicity during IL-2 therapy,inflammatory disorders, inflammation of autoimmune diseases, Crohn'sdisease, adult respiratory distress syndrome, thermal injury includingburns or frostbite, post-ischemic reperfusion conditions, myocardialinfarction, capillary leak syndrome, obesity, diabetes, Alzheimer'sdisease, schizophrenia, stroke, epilepsy, atherosclerosis, vasculitis,bullous pemphigoid, C3 glomerulopathy, membraneproliferativeglomerulonephritis, balloon angioplasty, post-pump syndrome incardiopulmonary bypass or renal bypass, hemodialysis, renal ischemia,mesenteric artery reperfusion after aortic reconstruction, infectiousdisease or sepsis, immune complex disorders and autoimmune diseases,diabetic nephropathy, Alport's syndrome, progressive kidney failure,proteinuric kidney diseases, renal ischemia-reperfusion injury, lupusnephritis, glomerulopathy, rheumatoid arthritis, systemic lupuserythematosus (SLE), SLE nephritis, membrano-proliferative nephritis,hemolytic anemia, neuromyelitis optica, renal transplant, inherited CD59deficiency, psoriasis, and myasthenia gravis. In certain otherembodiments, the antibodies of the present invention are useful fortreating or preventing at least one symptom or indication of aC5-associated disease or disorder selected from the group consisting oflung disease and disorders such as dyspnea, hemoptysis, ARDS, asthma,chronic obstructive pulmonary disease (COPD), emphysema, pulmonaryembolisms and infarcts, pneumonia, fibrogenic dust diseases, injury dueto inert dusts and minerals (e.g., silicon, coal dust, beryllium, andasbestos), pulmonary fibrosis, organic dust diseases, chemical injury(due to irritant gasses and chemicals, e.g., chlorine, phosgene, sulfurdioxide, hydrogen sulfide, nitrogen dioxide, ammonia, and hydrochloricacid), smoke injury, thermal injury (e.g., burn, freeze), asthma,allergy, bronchoconstriction, hypersensitivity pneumonitis, parasiticdiseases, Goodpasture's Syndrome, pulmonary vasculitis, hereditaryangioedema, and immune complex-associated inflammation.

In certain embodiments, the antibodies of the invention are useful totreat subjects suffering from an ocular disease such as age-relatedmacular degeneration (AMD), diabetic macular edema (DME), diabeticretinopathy, ocular angiogenesis (ocular neovascularization affectingchoroidal, corneal or retinal tissue), geographic atrophy (GA), uveitisand neuromyelitis optica. The antibodies of the present invention may beused to treat or to ameliorate at least one symptom or indication of dryAMD or wet AMD. In some embodiments, the antibodies of the invention areuseful in preventing or slowing rate of loss of vision. In oneembodiment, the antibodies of the present invention are useful inreducing drusen in the eye of a subject with dry AMD. In one embodiment,the antibodies of the present invention are useful in preventing orreducing/slowing loss of vision in a subject with AMD.

One or more antibodies of the present invention may be administered torelieve or prevent or decrease the severity of one or more of thesymptoms or conditions/indications of the ocular disease or disorder.The antibodies may be used to ameliorate or reduce the severity of atleast one symptom including, but not limited to loss of vision, visualdistortion, difficulty adapting to low light levels, crooked centralvision, increase in haziness of central/overall vision, presence ofdrusen (tiny accumulations of extracellular material that build up onthe retina), pigmentary changes, distorted vision in the form ofmetamorphopsia, in which a grid of straight lines appears wavy and partsof the grid may appear blank, exudative changes (hemorrhages in the eye,hard exudates, subretinal/sub-RPE/intraretinal fluid), slow recovery ofvisual function after exposure to bright light (photostress test),incipient and geographic atrophy, visual acuity drastically decreasing(two levels or more), e.g., 20/20 to 20/80, preferential hyperacuityperimetry changes (for wet AMD), blurred vision, gradual loss of centralvision (for those with non-exudative macular degeneration, rapid onsetof vision loss (often caused by leakage and bleeding of abnormal bloodvessels in subjects with exudative macular degeneration, centralscotomas (shadows or missing areas of vision), trouble discerningcolors, specifically dark ones from dark ones and light ones from lightones, loss in contrast sensitivity, straight lines appear curved in anAmsler grid.

It is also contemplated herein to use one or more antibodies of thepresent invention prophylactically to subjects at risk for developingmacular degeneration such as subjects over the age of 50, subjects witha family history of macular degeneration, smokers, and subjects withobesity, high cholesterol, cardiovascular disease, or unhealthy diet.

In a further embodiment of the invention the present antibodies are usedfor the preparation of a pharmaceutical composition or medicament fortreating patients suffering from a disease or disorder associated withC5. In another embodiment of the invention, the present antibodies areused as adjunct therapy with any other agent or any other therapy knownto those skilled in the art useful for treating or ameliorating adisease or disorder associated with C5.

Combination Therapies

Combination therapies may include an anti-C5 antibody of the inventionand any additional therapeutic agent that may be advantageously combinedwith an antibody of the invention, or with a biologically activefragment of an antibody of the invention. The antibodies of the presentinvention may be combined synergistically with one or more drugs ortherapy used to treat a disease or disorder associated with C5. In someembodiments, the antibodies of the invention may be combined with asecond therapeutic agent to ameliorate one or more symptoms of saiddisease.

Depending upon the C5-associated disease or disorder, the antibodies ofthe present invention may be used in combination with one or moreadditional therapeutic agents including, but not limited to, ananti-coagulant (e.g., warfarin, aspirin, heparin, phenindione,fondaparinux, idraparinux, and thrombin inhibitors such as argatroban,lepirudin, bivalirudin, or dabigatran) an anti-inflammatory drug (e.g.,corticosteroids, and non-steroidal anti-inflammatory drugs), anantihypertensive (e.g., an angiotensin-converting enzyme inhibitor), animmunosuppressive agent (e.g., vincristine, cyclosporine A, ormethotrexate), a fibrinolytic agent (e.g., ancrod, ε-aminocaproic acid,antiplasmin-a₁, prostacyclin, and defibrotide), a lipid-lowering agentsuch as an inhibitor of hydroxymethylglutaryl CoA reductase, ananti-CD20 agent such as rituximab, an anti-TNF agent such as infliximab,an anti-seizure agent (e.g., magnesium sulfate), a C3 inhibitor, or ananti-thrombotic agent.

In certain embodiments, the second therapeutic agent is another antibodyto C5 protein. It is contemplated herein to use a combination(“cocktail”) of antibodies with broad neutralization or inhibitoryactivity against C5. In some embodiments, non-competing antibodies maybe combined and administered to a subject in need thereof. In someembodiments, the antibodies comprising the combination bind to distinctnon-overlapping epitopes on the protein. The antibodies comprising thecombination may block the C5 binding to C5 convertase and/or mayprevent/inhibit cleavage of C5 into C5a and C5b. In certain embodiments,the second antibody may possess longer half-life in human serum.

As used herein, the term “in combination with” means that additionaltherapeutically active component(s) may be administered prior to,concurrent with, or after the administration of the anti-C5 antibody ofthe present invention. The term “in combination with” also includessequential or concomitant administration of an anti-C5 antibody and asecond therapeutic agent.

The additional therapeutically active component(s) may be administeredto a subject prior to administration of an anti-C5 antibody of thepresent invention. For example, a first component may be deemed to beadministered “prior to” a second component if the first component isadministered 1 week before, 72 hours before, 60 hours before, 48 hoursbefore, 36 hours before, 24 hours before, 12 hours before, 6 hoursbefore, 5 hours before, 4 hours before, 3 hours before, 2 hours before,1 hour before, 30 minutes before, 15 minutes before, 10 minutes before,5 minutes before, or less than 1 minute before administration of thesecond component. In other embodiments, the additional therapeuticallyactive component(s) may be administered to a subject afteradministration of an anti-C5 antibody of the present invention. Forexample, a first component may be deemed to be administered “after” asecond component if the first component is administered 1 minute after,5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after,6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hoursafter, 60 hours after, 72 hours after administration of the secondcomponent. In yet other embodiments, the additional therapeuticallyactive component(s) may be administered to a subject concurrent withadministration of an anti-C5 antibody of the present invention.“Concurrent” administration, for purposes of the present invention,includes, e.g., administration of an anti-C5 antibody and an additionaltherapeutically active component to a subject in a single dosage form,or in separate dosage forms administered to the subject within about 30minutes or less of each other. If administered in separate dosage forms,each dosage form may be administered via the same route (e.g., both theanti-C5 antibody and the additional therapeutically active component maybe administered intravenously, etc.); alternatively, each dosage formmay be administered via a different route (e.g., the anti-C5 antibodymay be administered intravenously, and the additional therapeuticallyactive component may be administered orally). In any event,administering the components in a single dosage from, in separate dosageforms by the same route, or in separate dosage forms by different routesare all considered “concurrent administration,” for purposes of thepresent disclosure. For purposes of the present disclosure,administration of an anti-C5 antibody “prior to”, “concurrent with,” or“after” (as those terms are defined herein above) administration of anadditional therapeutically active component is considered administrationof an anti-C5 antibody “in combination with” an additionaltherapeutically active component.

The present invention includes pharmaceutical compositions in which ananti-C5 antibody of the present invention is co-formulated with one ormore of the additional therapeutically active component(s) as describedelsewhere herein.

Administration Regimens

According to certain embodiments, a single dose of an anti-C5 antibodyof the invention (or a pharmaceutical composition comprising acombination of an anti-C5 antibody and any of the additionaltherapeutically active agents mentioned herein) may be administered to asubject in need thereof. According to certain embodiments of the presentinvention, multiple doses of an anti-C5 antibody (or a pharmaceuticalcomposition comprising a combination of an anti-C5 antibody and any ofthe additional therapeutically active agents mentioned herein) may beadministered to a subject over a defined time course. The methodsaccording to this aspect of the invention comprise sequentiallyadministering to a subject multiple doses of an anti-C5 antibody of theinvention. As used herein, “sequentially administering” means that eachdose of anti-C5 antibody is administered to the subject at a differentpoint in time, e.g., on different days separated by a predeterminedinterval (e.g., hours, days, weeks or months). The present inventionincludes methods which comprise sequentially administering to thepatient a single initial dose of an anti-C5 antibody, followed by one ormore secondary doses of the anti-C5 antibody, and optionally followed byone or more tertiary doses of the anti-C5 antibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the anti-C5 antibody ofthe invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount of anti-C5antibody, but generally may differ from one another in terms offrequency of administration. In certain embodiments, however, the amountof anti-C5 antibody contained in the initial, secondary and/or tertiarydoses varies from one another (e.g., adjusted up or down as appropriate)during the course of treatment. In certain embodiments, two or more(e.g., 2, 3, 4, or 5) doses are administered at the beginning of thetreatment regimen as “loading doses” followed by subsequent doses thatare administered on a less frequent basis (e.g., “maintenance doses”).

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 48 hours (e.g., 1,1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11,11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19,19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, ormore) after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of anti-C5 antibody which is administered to apatient prior to the administration of the very next dose in thesequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-C5 antibody. For example, in certain embodiments, only asingle secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In certain embodiments of the invention, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

Diagnostic Uses of the Antibodies

The anti-C5 antibodies of the present invention may be used to detectand/or measure C5 in a sample, e.g., for diagnostic purposes. Someembodiments contemplate the use of one or more antibodies of the presentinvention in assays to detect a C5-associated-disease or disorder.Exemplary diagnostic assays for C5 may comprise, e.g., contacting asample, obtained from a patient, with an anti-C5 antibody of theinvention, wherein the anti-C5 antibody is labeled with a detectablelabel or reporter molecule or used as a capture ligand to selectivelyisolate C5 from patient samples. Alternatively, an unlabeled anti-C5antibody can be used in diagnostic applications in combination with asecondary antibody which is itself detectably labeled. The detectablelabel or reporter molecule can be a radioisotope, such as ³H, ¹⁴C, ³²P,³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent moiety such asfluorescein isothiocyanate, or rhodamine; or an enzyme such as alkalinephosphatase, β-galactosidase, horseradish peroxidase, or luciferase.Specific exemplary assays that can be used to detect or measure C5 in asample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in C5 diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient, which contains detectable quantities of either C5 protein, orfragments thereof, under normal or pathological conditions. Generally,levels of C5 protein in a particular sample obtained from a healthypatient (e.g., a patient not afflicted with a disease associated withC5) will be measured to initially establish a baseline, or standard,level of C5. This baseline level of C5 can then be compared against thelevels of C5 measured in samples obtained from individuals suspected ofhaving a C5-associated condition, or symptoms associated with suchcondition.

The antibodies specific for C5 protein may contain no additional labelsor moieties, or they may contain an N-terminal or C-terminal label ormoiety. In one embodiment, the label or moiety is biotin. In a bindingassay, the location of a label (if any) may determine the orientation ofthe peptide relative to the surface upon which the peptide is bound. Forexample, if a surface is coated with avidin, a peptide containing anN-terminal biotin will be oriented such that the C-terminal portion ofthe peptide will be distal to the surface.

Selected Embodiments

Selected embodiments of the present disclosure include the following:

In Embodiment 1, the present invention includes an isolated antibody orantigen-binding fragment thereof that binds specifically to complementfactor 5 (C5) protein, wherein the antibody or antigen-binding fragmentthereof interacts with one or more amino acids contained within C5 (SEQID NO: 359), as determined by hydrogen/deuterium exchange.

In Embodiment 2, the present invention includes the isolated antibody ofantigen-binding fragment of Embodiment 1, wherein the antibody orantigen-binding fragment thereof interacts with one or more amino acidscontained within the alpha chain and/or the beta chain of 05, asdetermined by hydrogen/deuterium exchange.

In Embodiment 3, the present invention includes the isolated antibody ofantigen-binding fragment of Embodiments 1 or 2, wherein the antibody orantigen-binding fragment thereof does not interact with an amino acid ofthe C5a anaphylatoxin region of C5, as determined by hydrogen/deuteriumexchange.

In Embodiment 4, the present invention includes the isolated antibody ofantigen-binding fragment of any one of Embodiments 1 to 3, wherein theantibody or antigen-binding fragment thereof interacts with one or moreamino acids contained within SEQ ID NO: 360 and/or SEQ ID NO: 361, asdetermined by hydrogen/deuterium exchange.

In Embodiment 5, the present invention includes the isolated antibody orantigen-binding fragment of any one of Embodiments 1 to 4, wherein theantibody or antigen-binding fragment thereof interacts with an aminoacid sequence selected from the group consisting of (a) amino acids 591to 599 of SEQ ID NO: 359; (b) amino acids 593 to 599 of SEQ ID NO: 359;(c) amino acids 775 to 787 of SEQ ID NO: 359; (d) amino acids 775 to 794of SEQ ID NO: 359; and (e) amino acids 779 to 787 of SEQ ID NO: 359.

In Embodiment 6, the present invention includes the isolated antibody orantigen-binding fragment thereof of any one of Embodiments 1 to 5,wherein the antibody or antigen-binding fragment thereof interacts withat least five amino acids contained within an amino acid sequenceselected from the group consisting of SEQ ID NOs: 360 and 361.

In Embodiment 7, the present invention includes the isolated antibody orantigen-binding fragment thereof of any one of Embodiments 1 to 5,wherein the antibody or antigen-binding fragment thereof interacts withthe amino acid sequences of SEQ ID NOs: 360 and 361.

In Embodiment 8, the present invention includes an isolated antibody orantigen-binding fragment thereof that binds specifically to complementfactor 5 (C5) protein, wherein the antibody or antigen-binding fragmentthereof interacts with at least one of the following amino acidresidues: N591, M592, A593, T594, G595, M596, D597, S598, W599, W775,E776, V777, H778, L779, V780, P781, R782, R783, K784, Q785, L786, Q787,F788, A789, L790, P791, D792, S793, or L794 of SEQ ID NO: 359.

In Embodiment 9, the present invention includes the isolated antibody orantigen-binding fragment thereof of any one of Embodiments 1 to 8,wherein the antibody has one or more of the following characteristics:(a) has serum concentration of more than 10 μg/mL through day 70 uponadministration to cynomolgus monkey; (b) blocks classical pathway (CP)hemolysis through day 35 upon administration to cynomolgus monkey, asmeasured in an ex vivo hemolysis assay; (c) blocks alternative pathway(AP) hemolysis through day 35 upon administration to cynomolgus monkey,as measured in an ex vivo hemolysis assay; (d) has serum half-life ofmore than 10 days in cynomolgus monkey; (e) has serum concentration ofmore than 10 μg/mL through day 40 upon administration to C5-humanizedmice; (f) blocks CP hemolysis through day 30 upon administration toC5-humanized mice, as measured in an ex vivo hemolysis assay; and (g)has serum half-life of more than 10 days in C5-humanized mice.

In Embodiment 10, the present invention includes the isolated antibodyor antigen-binding fragment thereof of any one of Embodiments 1 to 9,wherein the antibody has an additional characteristic selected from thegroup consisting of: (a) is a fully human monoclonal antibody; (b) bindsto human C5 with a dissociation constant (K_(D)) of less than 0.9 nM at25° C., as measured in a surface plasmon resonance assay; (c) binds tohuman C5 with a K_(D) of less than 0.3 nM at 37° C., as measured in asurface plasmon resonance assay; (d) binds to monkey C5 with a K_(D) ofless than 65 nM, as measured in a surface plasmon resonance assay; (e)binds to human C5 variant R885H (SEQ ID NO: 356) with a K_(D) of lessthan 0.5 nM, as measured in a surface plasmon resonance assay; (f) bindsto human C5 variant R885C (SEQ ID NO: 357) with a K_(D) of less than 0.5nM, as measured in a surface plasmon resonance assay; (g) blocks humanC5-mediated classical pathway (CP) hemolysis by more than 95% and withIC₅₀ less than 6 nM, as measured in a CP hemolysis assay; (h) blockshuman C5-mediated alternative pathway (AP) hemolysis by more than 70%and with IC₅₀ less than 165 nM, as measured in a AP hemolysis assay; (i)inhibits African green monkey C5-mediated CP hemolysis with IC₅₀ lessthan 185 nM, as measured in a CP hemolysis assay; (j) inhibits Africangreen monkey C5-mediated AP hemolysis with IC₅₀ less than 235 nM, asmeasured in a AP hemolysis assay; (k) inhibits cynomolgus monkeyC5-mediated CP hemolysis with IC₅₀ less than 145 nM, as measured in a CPhemolysis assay; and (I) inhibits cynomolgus monkey C5-mediated APhemolysis with IC₅₀ less than 30 nM, as measured in a AP hemolysisassay.

In Embodiment 11, the present invention includes the isolated antibodyor antigen-binding fragment of any one of Embodiments 1 to 10, whereinthe antibody or antigen-binding fragment comprises three heavy chaincomplementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3)contained within any one of the heavy chain variable region (HCVR)sequences listed in Table 1; and three light chain CDRs (LCDR1, LCDR2and LCDR3) contained within any one of the light chain variable region(LCVR) sequences listed in Table 1.

In Embodiment 12, the present invention includes the isolated antibodyor antigen-binding fragment thereof of any one of Embodiments 1 to 11,comprising: (a)a HCDR1 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100,124, 140, 148, 156, 172, 188, 204, 220, 236, 252, 268, 276, 292, 308,324, and 340; (b)a HCDR2 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102,126, 142, 150, 158, 174, 190, 206, 222, 238, 254, 270, 278, 294, 310,326, and 342; (c) a HCDR3 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104,128, 144, 152, 160, 176, 192, 208, 224, 240, 256, 272, 280, 296, 312,328, and 344; (d)a LCDR1 domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108,116, 132, 164, 180, 196, 212, 228, 244, 260, 284, 300, 316, 332, and348; (e)a LCDR2 domain having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 118, 134,166, 182, 198, 214, 230, 246, 262, 286, 302, 318, 334, and 350; and (f)aLCDR3 domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 120, 136, 168,184, 200, 216, 232, 248, 264, 288, 304, 320, 336, and 352.

In Embodiment 13, the present invention includes the isolated antibodyor antigen-binding fragment thereof of any one of Embodiments 1 to 12comprising a HCVR having an amino acid sequence selected from the groupconsisting of HCVR sequences listed in Table 1.

In Embodiment 14, the present invention includes an isolated antibody orantigen-binding fragment thereof of Embodiment 13 comprising a LCVRhaving an amino acid sequence selected from the group consisting of LCVRsequences listed in Table 1.

In Embodiment 15, the present invention includes the isolated antibodyor antigen-binding fragment of any one of Embodiments 11 to 14comprising a HCVR/LCVR amino acid sequence pair selected from the groupconsisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90,98/106, 98/114, 122/106, 98/130, 138/106, 146/106, 122/130, 146/114,146/130, 138/130, 154/162, 170/178, 186/194, 202/210, 218/226, 234/242,250/258, 266/258, 274/282, 290/298, 306/314, 322/330, and 338/346.

In Embodiment 16, the present invention includes the isolated antibodyor antigen-binding fragment thereof of any one of Embodiments 11 to 15comprising three CDRs contained within a HCVR selected from the groupconsisting of SEQ ID NOs: 50, 98, 138, and 202; and three CDRs containedwithin a LCVR selected from the group consisting of SEQ ID NOs: 58, 106,and 210.

In Embodiment 17, the present invention includes the isolated antibodyor antigen-binding fragment thereof of Embodiment 16 comprising CDRsselected from the group consisting of: (a) SEQ ID NOs: 52, 54, 56, 60,62, and 64; (b) SEQ ID NOs: 100, 102, 104, 108, 110, and 112; (c) SEQ IDNOs: 140, 142, 144, 108, 110, and 112; and (d) SEQ ID NOs: 204, 206,208, 212, 214, and 216.

In Embodiment 18, the present invention includes the isolated antibodyor antigen-binding fragment thereof of Embodiment 17 comprising aHCVR/LCVR amino acid sequence pair selected from the group consisting ofSEQ ID NOs: 50/58, 98/106, 138/106, and 202/210.

In Embodiment 19, the present invention includes an antibody orantigen-binding fragment thereof that competes for binding to C5 withthe antibody or antigen-binding fragment thereof of Embodiment 17.

In Embodiment 20, the present invention includes an antibody orantigen-binding fragment thereof that binds to the same epitope as anantibody or antigen-binding fragment thereof of Embodiment 17.

In Embodiment 21, the present invention includes the antibody orantigen-binding fragment thereof of Embodiment 9 or 10 comprising aheavy chain variable region comprising an amino acid sequence listed inTable 1 having no more than 5 amino acid substitutions.

In Embodiment 22, the present invention includes the antibody orantigen-binding fragment thereof of Embodiment 21, comprising a lightchain variable region comprising an amino acid sequence listed in Table1 having no more than 5 amino acid substitutions.

In Embodiment 23, the present invention includes the antibody orantigen-binding fragment thereof of Embodiment 9 or 10 comprising aheavy chain variable region having at least 90% sequence identity to SEQID NO: 98.

In Embodiment 24, the present invention includes an antibody orantigen-binding fragment thereof of Embodiment 23 comprising a lightchain variable region having at least 90% sequence identity to SEQ IDNO: 106.

In Embodiment 25, the present invention includes an isolated monoclonalantibody or antigen-binding fragment thereof that blocks C5 cleavage toC5a and C5b comprising three CDRs of a HCVR, wherein the HCVR has anamino acid sequence selected from the group consisting of SEQ ID NOs: 2,18, 34, 50, 66, 82, 98, 122, 138, 146, 154, 170, 186, 202, 218, 234,250, 266, 274, 290, 306, 322, and 338; and three CDRs of a LCVR, whereinthe LCVR has an amino acid sequence selected from the group consistingof SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 114, 130, 162, 178, 194,210, 226, 242, 258, 282, 298, 314, 330, and 346.

In Embodiment 26, the present invention includes a pharmaceuticalcomposition comprising an isolated antibody or antigen-binding fragmentthereof that binds to C5 according to any one of Embodiments 1 to 25 anda pharmaceutically acceptable carrier or diluent.

In Embodiment 27, the present invention includes an isolatedpolynucleotide molecule comprising a polynucleotide sequence thatencodes a HCVR of an antibody as set forth in any one of Embodiments 1to 25.

In Embodiment 28, the present invention includes an isolatedpolynucleotide molecule comprising a polynucleotide sequence thatencodes a LCVR of an antibody as set forth in any one of Embodiments 1to 25.

In Embodiment 29, the present invention includes a vector comprising thepolynucleotide sequence of Embodiment 27 or 28.

In Embodiment 30, the present invention includes a cell expressing thevector of Embodiment 29.

In Embodiment 31, the present invention includes a method of preventing,treating or ameliorating at least one symptom or indication of a diseaseor disorder associated with C5, the method comprising administering anantibody or antigen-binding fragment of any one of Embodiments 1 to 25to a subject in need thereof.

In Embodiment 32, the present invention includes the method ofEmbodiment 31, wherein the disease or disorder is selected from thegroup consisting of atypical hemolytic uremic syndrome (aHUS),paroxysmal nocturnal hemoglobinuria (PNH), age-related maculardegeneration, geographic atrophy, uveitis, neuromyelitis optica,multiple sclerosis, stroke, Guillain Barre Syndrome, traumatic braininjury, Parkinson's disease, disorders of inappropriate or undesirablecomplement activation, hemodialysis complications, hyperacute allograftrejection, xenograft rejection, interleukin-2 induced toxicity duringIL-2 therapy, inflammatory disorders, inflammation of autoimmunediseases, Crohn's disease, adult respiratory distress syndrome, thermalinjury including burns or frostbite, post-ischemic reperfusionconditions, myocardial infarction, capillary leak syndrome, obesity,diabetes, Alzheimer's disease, schizophrenia, stroke, epilepsy,atherosclerosis, vasculitis, bullous pemphigoid, C3 glomerulopathy,membraneproliferative glomerulonephritis, diabetic nephropathy, Alport'ssyndrome, progressive kidney failure, proteinuric kidney diseases, renalischemia-reperfusion injury, lupus nephritis, balloon angioplasty,post-pump syndrome in cardiopulmonary bypass or renal bypass,hemodialysis, renal ischemia, mesenteric artery reperfusion after aorticreconstruction, infectious disease or sepsis, immune complex disordersand autoimmune diseases, renal disorders, rheumatoid arthritis, systemiclupus erythematosus (SLE), SLE nephritis, proliferative nephritis,hemolytic anemia, asthma, chronic obstructive pulmonary disease (COPD),emphysema, pulmonary embolisms and infarcts, pneumonia, and myastheniagravis.

In Embodiment 33, the present invention includes the method ofEmbodiment 31, wherein the disease or disorder is aHUS.

In Embodiment 34, the present invention includes the method ofEmbodiment 31, wherein the disease or disorder is PNH.

In Embodiment 35, the present invention includes the method of any oneof Embodiments 31-34, wherein the pharmaceutical composition isadministered prophylactically or therapeutically to the subject in needthereof.

In Embodiment 36, the present invention includes the method of any oneof Embodiments 31 to 35, wherein the pharmaceutical composition isadministered in combination with a second therapeutic agent.

In Embodiment 37, the present invention includes the method ofEmbodiment 36, wherein the second therapeutic agent is selected from thegroup consisting of an anti-coagulant, an anti-inflammatory drug, anantihypertensive, an immunosuppressive agent, a lipid-lowering agent, ananti-CD20 agent such as rituximab, an anti-TNF agent such as infliximab,an anti-seizure agent, a C3 inhibitor, a second anti-C5 antibody, and ananti-thrombotic agent.

In Embodiment 38, the present invention includes the method of any oneof Embodiments 31 to 37, wherein the pharmaceutical composition isadministered subcutaneously, intravenously, intradermally,intraperitoneally, orally, intramuscularly or intracranially.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, room temperatureis about 25° C., and pressure is at or near atmospheric.

Example 1: Generation of Human Antibodies to Complement Factor 5 (C5)Protein

Human antibodies to C5 protein were generated in a VELOCIMMUNE® mousecomprising DNA encoding human Immunoglobulin heavy and kappa light chainvariable regions. The mice were immunized with serum purified human C5protein (CALBIOCHEM® Cat #20-4888).

The antibody immune response was monitored by a C5-specific immunoassay.When a desired immune response was achieved splenocytes were harvestedand fused with mouse myeloma cells to preserve their viability and formhybridoma cell lines. The hybridoma cell lines were screened andselected to identify cell lines that produce C5-specific antibodies. Thecell lines were used to obtain several anti-C5 chimeric antibodies(i.e., antibodies possessing human variable domains and mouse constantdomains); exemplary antibodies generated in this manner were designatedas H2M11683N and H2M11686N.

Anti-C5 antibodies were also isolated directly from antigen-positivemouse B cells without fusion to myeloma cells, as described in U.S. Pat.No. 7,582,298, herein specifically incorporated by reference in itsentirety. Using this method, several fully human anti-C5 antibodies(i.e., antibodies possessing human variable domains and human constantdomains) were obtained; exemplary antibodies generated in this mannerwere designated as H4H12159P, H4H12161P, H4H12163P, H4H12164P,H4H12166P, H4H12167P, H4H12168P, H4H12169P, H4H12170P, H4H12171P,H4H12175P, H4H12176P2, H4H12177P2 and H4H12183P2.

The biological properties of the exemplary antibodies generated inaccordance with the methods of this Example are described in detail inthe Examples set forth below.

Example 2: Heavy and Light Chain Variable Region Amino Acid andNucleotide Sequences

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-C5 antibodies ofthe invention.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H2M11683N 2 4 6 8 10 12 1416 H2M11686N 18 20 22 24 26 28 30 32 H4H12159P 34 36 38 40 42 44 46 48H4H12161P 50 52 54 56 58 60 62 64 H4H12163P 66 68 70 72 74 76 78 80H4H12164P 82 84 86 88 90 92 94 96 H4H12166P 98 100 102 104 106 108 110112 H4H12166P2 98 100 102 104 114 116 118 120 H4H12166P3 122 124 126 128106 108 110 112 H4H12166P4 98 100 102 104 130 132 134 136 H4H12166P5 138140 142 144 106 108 110 112 H4H12166P6 146 148 150 152 106 108 110 112H4H12166P7 122 124 126 128 130 132 134 136 H4H12166P8 146 148 150 152114 116 118 120 H4H12166P9 146 148 150 152 130 132 134 136 H4H12166P10138 140 142 144 130 132 134 136 H4H12167P 154 156 158 160 162 164 166168 H4H12168P 170 172 174 176 178 180 182 184 H4H12169P 186 188 190 192194 196 198 200 H4H12170P 202 204 206 208 210 212 214 216 H4H12171P 218220 222 224 226 228 230 232 H4H12175P 234 236 238 240 242 244 246 248H4H12176P2 250 252 254 256 258 260 262 264 H4H12177P2 266 268 270 272258 260 262 264 H4H12183P2 274 276 278 280 282 284 286 288 H2M11682N 290292 294 296 298 300 302 304 H2M11684N 306 308 310 312 314 316 318 320H2M11694N 322 324 326 328 330 332 334 336 H2M11695N 338 340 342 344 346348 350 352

The corresponding nucleic acid sequence identifiers are set forth inTable 2.

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H2M11683N 1 35 7 9 11 13 15 H2M11686N 17 19 21 23 25 27 29 31 H4H12159P 33 35 37 3941 43 45 47 H4H12161P 49 51 53 55 57 59 61 63 H4H12163P 65 67 69 71 7375 77 79 H4H12164P 81 83 85 87 89 91 93 95 H4H12166P 97 99 101 103 105107 109 111 H4H12166P2 97 99 101 103 113 115 117 119 H4H12166P3 121 123125 127 105 107 109 111 H4H12166P4 97 99 101 103 129 131 133 135H4H12166P5 137 139 141 143 105 107 109 111 H4H12166P6 145 147 149 151105 107 109 111 H4H12166P7 121 123 125 127 129 131 133 135 H4H12166P8145 147 149 151 113 115 117 119 H4H12166P9 145 147 149 151 129 131 133135 H4H12166P10 137 139 141 143 129 131 133 135 H4H12167P 153 155 157159 161 163 165 167 H4H12168P 169 171 173 175 177 179 181 183 H4H12169P185 187 189 191 193 195 197 199 H4H12170P 201 203 205 207 209 211 213215 H4H12171P 217 219 221 223 225 227 229 231 H4H12175P 233 235 237 239241 243 245 247 H4H12176P2 249 251 253 255 257 259 261 263 H4H12177P2265 267 269 271 257 259 261 263 H4H12183P2 273 275 277 279 281 283 285287 H2M11682N 289 291 293 295 297 299 301 303 H2M11684N 305 307 309 311313 315 317 319 H2M11694N 321 323 325 327 329 331 333 335 H2M11695N 337339 341 343 345 347 349 351

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H4H,” “H2M,” etc.), followed by anumerical identifier (e.g. “11686,” “12166,” “12183,” etc., as shown inTable 2), followed by a “P,” “P2,” or “N” suffix. Thus, according tothis nomenclature, an antibody may be referred to herein as, e.g.,“H2M11686N,” “H4H12183P2,” “H4H12168P,” etc. The H4H and H2M prefixes onthe antibody designations used herein indicate the particular Fc regionisotype of the antibody. For example, an “H4H” antibody has a human IgG4Fc comprising a serine to proline mutation in the hinge region (S108P)to promote dimer stabilization, and an “H2M” antibody has a mouse IgG2Fc (a or b isotype) (all variable regions are fully human as denoted bythe first ‘H’ in the antibody designation). As will be appreciated by aperson of ordinary skill in the art, an antibody having a particular Fcisotype can be converted to an antibody with a different Fc isotype(e.g., an antibody with a mouse IgG1 Fc can be converted to an antibodywith a human IgG4, etc.), but in any event, the variable domains(including the CDRs)—which are indicated by the numerical identifiersshown in Table 2—will remain the same, and the binding properties toantigen are expected to be identical or substantially similar regardlessof the nature of the Fc domain.

In certain embodiments, selected antibodies with a mouse IgG1 Fc wereconverted to antibodies with human IgG4 Fc. In one embodiment, the IgG4Fc domain comprises 2 or more amino acid changes as disclosed inUS20100331527.

To generate mutated antibodies, various residues in the complementarydetermining regions (CDRs) of H4H12166P were mutated to histidine togenerate 9 mutated antibodies, identified as H4H12166P2 to H4H12166P10.Histidine mutations in the CDRs have been shown to confer pH-dependenceof binding to target antigen leading to improved pharmacokinetics (Igawaet al. 2010, Nat. Biotechnol. 28: 1203-1207).

Control Constructs Used in the Following Examples

The following control constructs (anti-C5 antibodies) were included inthe experiments disclosed herein, for comparative purposes: “Comparator1,” a monoclonal antibody against human C5 having V_(H)/V_(L) sequencesof antibody “h5G1.1” according to U.S. Pat. No. 6,355,245 (AlexionPharmaceuticals, Inc.); and “Comparator 2,” a human monoclonal antibodyagainst human C5 having V_(H)/V_(L) sequences of antibody “8109”according to US Patent Application Publication No. 2013/0022615(Novartis).

Example 3: Antibody Binding to C5 as Determined by Surface PlasmonResonance

Equilibrium dissociation constants (K_(D) values) for C5 binding topurified anti-C5 antibodies were determined using a real-time surfaceplasmon resonance biosensor assay on a BIACORE™ T200 instrument. TheBIACORE™sensor surface was derivatized by amine coupling with amonoclonal mouse anti-human Fc antibody (GE Healthcare, # BR-1008-39) tocapture anti-C5 antibodies expressed with human Fc constant regions.BIACORE™binding studies were conducted in HBST running buffer (0.01MHEPES pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20). Human C5was obtained from a commercial source (EMD). Other C5 reagents wereexpressed with a C-terminal myc-myc-hexahistidine tag (subsequentlyreferred to as C5-mmh). Human C5-mmh reagents were also expressedcontaining histidine and cysteine point mutations at arginine 885(subsequently referred to as C5 R885H-mmh and C5 R885C-mmh,respectively). Different concentrations of human C5, human C5 R885H-mmh(SEQ ID No: 356), human C5 R885C-mmh (SEQ ID No: 357) and monkey C5-mmh(SEQ ID No: 358) (ranging from 100 nM to 1.23 nM, 3-fold dilutions)prepared in HBST running buffer were injected over the anti-C5 antibodycaptured surface at a flow rate of 30 μL/min. Association of all the C5reagents to each of the captured monoclonal antibodies was monitored for3 minutes and their dissociation in HBST running buffer was monitoredfor 8 minutes. All the binding kinetics experiments were performed ateither 25° C. or 37° C. Kinetic association (k_(a)) and dissociation(k_(d)) rate constants were determined by fitting the real-timesensorgrams to a 1:1 binding model using Scrubber 2.0c curve fittingsoftware. Binding dissociation equilibrium constants (K_(D)) anddissociative half-lives (t½) were calculated from the kinetic rateconstants as: K_(D) (M)=k_(d)/k_(a) and t½ (min)=In2/(60×k_(d))

Binding kinetic parameters for human C5 binding to anti-C5 antibodies at25° C. and 37° C. are shown in Tables 3 and 4.

TABLE 3 Binding Kinetics parameters of anti-C5 monoclonal antibodiesbinding to human C5 at 25° C. Amount of 100 nM Antibody Human C5Captured Bound k_(a) k_(d) KD t½ Antibody (RU) (RU) (1/Ms) (1/s) (M)(min) H4H11683N 231 439 4.17E+05 5.14E−05 1.23E−10 225 H4H12171P 51 641.49E+05 8.16E−05 5.49E−10 142 H4H12161P 38 64 2.58E+05 4.37E−051.70E−10 264 H4H12176P2 50 96 3.36E+05 6.75E−05 2.01E−10 171 H4H12163P51 108 6.43E+05 2.08E−04 3.24E−10 55 H4H12167P 52 116 1.09E+06 1.31E−041.21E−10 88 H4H12175P 51 100 2.16E+05 1.96E−04 9.10E−10 59 H4H12159P 53118 9.75E+05 7.13E−05 7.31E−11 162 H4H12164P 52 103 2.92E+05 8.84E−053.02E−10 131 H4H12168P 50 113 4.23E+05 4.75E−05 1.12E−10 243 H4H12169P51 18 2.24E+05 4.40E−04 1.96E−09 26 H4H11686N 200 341 2.20E+05 3.31E−051.50E−10 349 H4H12170P 51 119 5.25E+05 6.79E−05 1.29E−10 170 H4H12177P247 60 6.56E+04 6.29E−05 9.59E−10 184 H4H12183P2 46 50 1.66E+05 2.70E−051.63E−10 427 H4H12166P 53 105 6.42E+05 1.10E−04 1.71E−10 105 H4H12166P253 95 8.26E+05 3.61E−04 4.38E−10 32 H4H12166P3 59 124 4.03E+05 4.65E−041.15E−09 25 H4H12166P4 49 92 4.46E+05 1.76E−04 3.95E−10 66 H4H12166P5 59110 2.85E+05 3.28E−04 1.15E−09 35 H4H12166P6 64 131 4.89E+05 1.84E−043.75E−10 63 H4H12166P7 50 92 2.74E+05 1.01E−03 3.67E−09 11 H4H12166P8 5091 4.84E+05 6.86E−04 1.42E−09 17 H4H12166P9 52 100 3.32E+05 2.64E−047.94E−10 44 H4H12166P10 49 69 1.57E+05 1.32E−03 8.38E−09 9 Comparator 1232 250 9.69E+04 1.46E−04 1.51E−09 79 Comparator 2 117 170 2.62E+052.39E−04 9.12E−10 48

TABLE 4 Binding Kinetics parameters of anti-C5 monoclonal antibodiesbinding to human C5 at 37° C. Amount of 100 nM Antibody Human C5Captured Bound k_(a) k_(d) K_(D) t½ Antibody (RU) (RU) (1/Ms) (1/s) (M)(min) H4H11683N 257 492 4.54E+05 2.41E−04 5.32E−10 48 H4H12171P 59 581.22E+05 7.62E−04 6.27E−09 15 H4H12161P 40 66 1.16E+05 1.15E−04 9.90E−10101 H4H12176P2 38 71 1.47E+05 2.34E−04 1.59E−09 49 H4H12163P 65 1399.11E+05 6.65E−04 7.29E−10 17 H4H12167P 75 153 1.29E+06 3.81E−042.95E−10 30 H4H12175P 74 132 2.96E+05 6.37E−04 2.15E−09 18 H4H12159P 70145 1.04E+06 1.07E−04 1.03E−10 108 H4H12164P 66 140 3.96E+05 1.28E−043.23E−10 90 H4H12168P 34 12 2.50E+04 4.64E−04 1.85E−08 25 H4H12169P 5965 1.15E+05 3.52E−04 3.06E−09 33 H4H11686N 206 406 3.33E+05 1.56E−044.69E−10 74 H4H12170P 34 55 2.97E+05 4.15E−04 1.40E−09 28 H4H12177P2 4137 4.42E+04 5.78E−04 1.31E−08 20 H4H12183P2 29 30 4.30E+04 2.50E−045.81E−09 46 H4H12166P 69 127 8.80E+05 2.30E−04 2.62E−10 50 H4H12166P2 68110 9.50E+05 1.23E−03 1.29E−09 9 H4H12166P3 86 147 6.12E+05 1.27E−032.07E−09 9 H4H12166P4 63 108 5.05E+05 4.69E−04 9.30E−10 25 H4H12166P5 76129 4.40E+05 1.22E−03 2.77E−09 9 H4H12166P6 90 157 5.42E+05 4.74E−048.75E−10 24 H4H12166P7 64 105 3.49E+05 2.58E−03 7.39E−09 4 H4H12166P8 6598 6.75E+05 2.09E−03 3.10E−09 6 H4H12166P9 76 122 3.75E+05 6.39E−041.70E−09 18 H4H12166P10 64 82 2.27E+05 3.14E−03 1.38E−08 4 Comparator 1185 246 1.47E+05 5.30E−04 3.61E−09 22 Comparator 2 119 205 2.85E+056.57E−04 2.30E−10 18

Monkey C5-mmh binding to anti-C5 antibodies at 25° C. and 37° C. areshown in Tables 5 and 6.

TABLE 5 Binding Kinetics parameters of anti-C5 monoclonal antibodiesbinding to monkey C5-mmh at 25° C. Amount 100 nM of monkey AntibodyC5-mmh Captured Bound ka kd K_(D) t 1/2 Antibody (RU) (RU) (1/Ms) (1/s)(M) (min) H4H11683N 228 403 3.86E+05 2.47E−04 6.40E−10 47 H4H12171P 5117 4.60E+04 2.26E−04 4.92E−09 51 H4H12161P 38 45 6.33E+04 2.48E−053.92E−10 465 H4H12176P2 50 69 1.82E+05 5.88E−05 3.22E−10 196 H4H12163P50 98 3.11E+05 7.75E−04 2.49E−09 15 H4H12167P 52 111 4.19E+05 1.32E−043.15E−10 88 H4H12175P 51 59 6.42E+04 1.65E−03 2.57E−08 7 H4H12159P 53116 3.54E+05 4.69E−05 1.33E−10 246 H4H12164P 51 66 1.27E+05 1.53E−031.20E−08 8 H4H12168P 50 86 1.73E+05 1.14E−04 6.60E−10 101 H4H12169P 5122 1.64E+05 4.55E−03 2.78E−08 3 H4H11686N 196 247 1.57E+05 4.89E−043.11E−09 24 H4H12170P 51 92 2.62E+05 5.21E−05 1.99E−10 222 H4H12177P2 4732 4.62E+04 9.92E−04 2.15E−08 12 H4H12183P2 47 23 4.88E+04 4.94E−041.01E−08 23 H4H12166P 52 90 2.05E+05 1.06E−03 5.15E−09 11 H4H12166P2 5371 3.00E+05 3.16E−03 1.05E−08 4 H4H12166P3 59 72 1.68E+05 4.47E−032.66E−08 3 H4H12166P4 49 69 2.10E+05 1.78E−03 8.50E−09 6 H4H12166P5 5956 1.44E+05 3.46E−03 2.40E−08 3 H4H12166P6 64 94 2.39E+05 2.66E−031.11E−08 4 H4H12166P7 50 36 1.36E+05 6.33E−03 4.65E−08 2 H4H12166P8 5047 2.31E+05 4.99E−03 2.16E−08 2 H4H12166P9 52 55 1.70E+05 3.18E−031.87E−08 4 H4H12166P10 49 15 9.56E+04 6.16E−03 6.44E−08 2 Comparator 1*228 11 N/A N/A 3.11E−07 N/A N/A = Not Available; *SS = steady stateanalysis

TABLE 6 Binding Kinetics parameters of anti-C5 monoclonal antibodiesbinding to monkey C5-mmh at 37° C. 100 nM Amount of monkey AntibodyC5-mmh Captured Bound ka kd KD t 1/2 Antibody (RU) (RU) (1/Ms) (1/s) (M)(min) H4H11683N 192 303 5.35E+05 1.15E−03 2.16E−09 10 H4H12171P 59 785.56E+05 1.03E−03 1.85E−09 11 H4H12161P 41 53 1.34E+05 7.45E−04 5.56E−0916 H4H12176P2 36 47 1.35E+05 1.29E−03 9.60E−09 9 H4H12163P 64 1293.90E+05 1.25E−03 3.20E−09 9 H4H12167P 74 146 5.37E+05 2.89E−04 5.39E−1040 H4H12175P 74 74 1.77E+05 2.76E−03 1.56E−08 4 H4H12159P 70 1374.12E+05 5.50E−05 1.33E−10 210 H4H12164P 65 99 1.86E+05 1.17E−036.30E−09 10 H4H12168P 34 29 5.33E+04 6.76E−04 1.27E−08 17 H4H12169P 5964 2.51E+05 3.61E−03 1.43E−08 3 H4H11686N 145 195 2.33E+05 2.07E−038.88E−09 6 H4H12170P 34 60 5.21E+05 8.71E−04 1.67E−09 13 H4H12177P2 4127 1.50E+05 7.17E−03 4.77E−08 2 H4H12183P2 28 13 5.40E+04 6.37E−031.18E−07 2 H4H12166P 68 110 2.19E+05 1.87E−03 8.55E−09 6 H4H12166P2 6883 3.93E+05 2.97E−03 7.56E−09 4 H4H12166P3 85 92 2.23E+05 2.92E−031.31E−08 4 H4H12166P4 62 80 2.23E+05 1.83E−03 8.20E−09 6 H4H12166P5 7570 1.50E+05 3.13E−03 2.09E−08 4 H4H12166P6 90 112 2.53E+05 2.32E−039.18E−09 5 H4H12166P7 63 48 1.25E+05 2.41E−03 1.93E−08 5 H4H12166P8 6453 2.03E+05 2.69E−03 1.33E−08 4 H4H12166P9 75 69 1.81E+05 2.61E−031.44E−08 4 H4H12166P10 63 24 6.60E+04 2.79E−03 4.22E−08 4 Comparator 1132 4 NB NB NB NB

Human C5 R885H-mmh and human C5 R885C-mmh binding to anti-C5 antibodiesat 25° C. are shown in Tables 7 and 8, respectively.

TABLE 7 Binding Kinetics parameters of anti-C5 monoclonal antibodiesbinding to human C5 R885H-mmh at 25° C. 100 nM Amount of Human C5Antibody R885H-mmh Captured Bound k_(a) k_(d) K_(D) t½ Antibody (RU)(RU) (1/Ms) (1/s) (M) (min) H4H11683N 183 118 5.26E+05 2.46E−04 4.68E−1047 H4H12171P 119 51 6.59E+05 1.42E−04 2.16E−10 81 H4H12161P 105 1998.36E+04 8.32E−05 9.96E−10 139 H4H12176P2 170 65 1.78E+05 2.17E−041.22E−09 53 H4H12163P 111 214 6.72E+05 4.34E−04 6.46E−10 27 H4H12167P 93187 6.89E+05 2.98E−04 4.33E−10 39 H4H12175P 104 207 1.81E+05 1.98E−031.09E−08 6 H4H12159P 101 177 7.06E+05 1.76E−04 2.50E−10 66 H4H12164P 143295 1.58E+05 1.87E−04 1.19E−09 62 H4H12168P 138 197 5.29E+04 2.14E−044.05E−09 54 H4H12169P 116 173 4.84E+05 7.09E−05 1.47E−10 163 H4H11686N145 259 2.16E+05 1.06E−04 4.91E−10 109 H4H12170P 244 442 4.09E+051.61E−04 3.94E−10 72 H4H12177P2 137 232 1.48E+05 5.92E−04 4.01E−09 20H4H12183P2 158 99 3.77E+04 4.37E−05 1.16E−09 264 H4H12166P 188 3665.28E+05 2.12E−04 4.02E−10 54 Comparator 1 87 11 NB NB NB NB Comparator2 118 249 1.08E+06 6.53E−04 6.06E−10 18

TABLE 8 Binding Kinetics parameters of anti-C5 monoclonal antibodiesbinding to human C5 R885C-mmh at 25° C. 100 nM Amount of Human C5Antibody R885C-mmh Captured Bound k_(a) k_(d) K_(D) t½ Antibody (RU)(RU) (1/Ms) (1/s) (M) (min) H4H11683N 174 116 4.99E+05 2.39E−04 4.79E−1048 H4H12171P 109 51 3.79E+05 1.39E−04 3.66E−10 83 H4H12161P 103 1471.30E+05 8.71E−05 6.72E−10 133 H4H12176P2 164 63 1.07E+05 2.18E−042.03E−09 53 H4H12163P 110 211 5.04E+05 4.32E−04 8.58E−10 27 H4H12167P 85163 7.11E+05 2.94E−04 4.13E−10 39 H4H12175P 99 128 8.18E+04 1.55E−021.90E−07 1 H4H12159P 93 168 5.86E+05 1.69E−04 2.89E−10 68 H4H12164P 139249 1.53E+05 1.82E−04 1.19E−09 63 H4H12168P 128 144 6.09E+04 1.99E−043.27E−09 58 H4H12169P 108 168 2.78E+05 6.99E−05 2.51E−10 165 H4H11686N143 253 1.78E+05 9.49E−05 5.34E−10 122 H4H12170P 244 427 3.57E+051.60E−04 4.47E−10 72 H4H12177P2 138 177 1.00E+05 1.32E−03 1.32E−08 9H4H12183P2 158 80 2.99E+04 2.20E−05 7.37E−10 525 H4H12166P 188 3564.26E+05 2.07E−04 4.87E−10 56 Comparator 1 87 9 NB NB NB NB Comparator 2117 241 1.17E+06 6.19E−04 5.30E−10 19

Human C5 R885H-mmh and human C5 R885C-mmh binding to anti-C5 antibodiesat 37° C. are shown in Tables 9 and 10, respectively.

TABLE 9 Binding Kinetics parameters of anti-C5 monoclonal antibodiesbinding to human C5 R885H-mmh at 37° C. 100 nM Amount of Human C5Antibody R885H-mmh Captured Bound k_(a) k_(d) K_(D) t½ Antibody (RU)(RU) (1/Ms) (1/s) (M) (min) H4H11683N 49 81 5.48E+05 1.47E−03 2.69E−09 8H4H12171P 59 80 5.92E+05 9.63E−04 1.63E−09 12 H4H12161P 41 54 1.18E+059.25E−04 7.84E−09 12 H4H12176P2 45 69 2.57E+05 9.58E−04 3.73E−09 12H4H12163P 60 85 7.24E+05 2.90E−03 4.00E−09 4 H4H12167P 38 65 8.81E+052.57E−03 2.91E−09 5 H4H12175P 25 30 1.37E+05 9.50E−03 6.94E−08 1H4H12159P 51 82 6.38E+05 9.48E−04 1.49E−09 12 H4H12164P 59 68 1.95E+051.06E−03 5.46E−09 11 H4H12168P 34 29 2.43E+04 1.23E−03 5.04E−08 9H4H12169P 61 79 4.29E+05 7.39E−04 1.72E−09 16 H4H11686N 40 74 4.19E+058.00E−04 1.91E−09 14 H4H12170P 36 64 5.59E+05 8.39E−04 1.50E−09 14H4H12177P2 45 51 2.76E+05 2.76E−03 1.00E−08 4 H4H12183P2 33 36 9.58E+047.12E−04 7.43E−09 16 H4H12166P 71 58 6.24E+05 1.31E−03 2.09E−09 9Comparator 1 41 5 NB NB NB NB Comparator 2 23 47 8.39E+05 1.05E−031.25E−09 11

TABLE 10 Binding Kinetics parameters of anti-C5 monoclonal antibodiesbinding to human C5 R885C-mmh at 37° C. 100 nM Amount of Human C5Antibody R885C-mmh Captured Bound k_(a) k_(d) K_(D) t½ Antibody (RU)(RU) (1/Ms) (1/s) (M) (min) H4H11683N 48 78 4.38E+05 1.43E−03 3.25E−09 8H4H12171P 59 78 4.77E+05 9.57E−04 2.01E−09 12 H4H12161P 41 49 1.10E+059.01E−04 8.17E−09 13 H4H12176P2 44 55 1.41E+05 1.03E−03 7.32E−09 11H4H12163P 59 83 5.66E+05 2.81E−03 4.97E−09 4 H4H12167P 38 64 6.84E+052.49E−03 3.64E−09 5 H4H12175P 25 4 1.12E+05 1.79E−02 1.59E−07 1H4H12159P 51 68 5.61E+05 9.75E−04 1.74E−09 12 H4H12164P 59 64 1.77E+051.04E−03 5.85E−09 11 H4H12168P 34 21 6.38E+04 5.69E−04 8.90E−09 20H4H12169P 61 75 3.29E+05 7.37E−04 2.24E−09 16 H4H11686N 39 69 2.84E+057.91E−04 2.78E−09 15 H4H12170P 36 61 4.24E+05 8.70E−04 2.05E−09 13H4H12177P2 43 31 1.07E+05 5.07E−03 4.76E−08 2 H4H12183P2 31 25 5.12E+049.97E−04 1.95E−08 12 H4H12166P 72 54 4.91E+05 1.26E−03 2.56E−09 9Comparator 1 41 2 NB NB NB NB Comparator 2 23 42 7.34E+05 1.07E−031.45E−09 11

At 25° C., all 25 anti-C5 antibodies of the invention bound to human C5with K_(D) values ranging from 73 pM to 8.4 nM as shown in Table 3. At37° C., the anti-C5 antibodies of the invention bound to human C5 withK_(D) values ranging from 103 pM to 18.5 nM as shown in Table 4. At 25°C., 25 out of the 25 anti-C5 antibodies of the invention tested bound tomonkey C5-mmh with K_(D) values ranging from 133 pM to 64 nM as shown inTable 5. At 37° C., 25 out of the 25 anti-C5 antibodies of the inventiontested bound to monkey C5-mmh with K_(D) values ranging from 133 pM to118 nM as shown in Table 6. At 25° C., 16 out of the 16 anti-C5antibodies of the invention tested bound to human C5 R885H-mmh withK_(D) values ranging from 147 pM to 10.9 nM as shown in Table 7. At 25°C., 16 out of the 16 anti-C5 antibodies of the invention tested bound tohuman C5 R885C-mmh with K_(D) values ranging from 251 pM to 190 nM asshown in Table 8. At 37° C., 16 out of the 16 anti-C5 antibodies of theinvention tested bound to human C5 R885H-mmh with K_(D) values rangingfrom 1.49 nM to 69.4 nM as shown in Table 9. At 25° C., 16 out of the 16anti-C5 antibodies of the invention tested bound to human C5 R885C-mmhwith K_(D) values ranging from 1.74 nM to 159 nM as shown in Table 10.

Example 4: Antibody Binding to C5 Through Different pH

Effect of pH on the rate of dissociation of recombinant human C5 boundto purified anti-C5 monoclonal antibodies was determined using areal-time surface plasmon resonance biosensor using a BIACORE™T200instrument. The BIACORE™sensor surface was first derivatized by aminecoupling with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to capture anti-C5 monoclonal antibodies expressed withhuman IgG4 Fc. All BIACORE™binding studies were performed using tworunning buffers PBS-T, pH7.4 (0.01M Na₂HPO₄/NaH₂PO₄, 0.15M NaCl, 0.05%v/v Tween-20, adjusted to pH7.4) and PBS-T, pH6.0 (0.01MNa₂HPO₄/NaH₂PO₄, 0.15M NaCl, 0.05% v/v Tween-20, adjusted to pH6.0).Different concentrations of human C5 (EMD, Catalog #204888) or monkeyC5.mmh (prepared in PBS-T, pH7.4 running buffer (ranging from 100 nM to11.11 nM, 3-fold dilutions) were injected over the anti-C5 monoclonalantibody captured surface for 3 minutes at a flow rate of 50 μL/minuteand their dissociation in two running buffers PBS-T, pH7.4 and PBS-T,pH6.0 was monitored for 6 minutes. All the binding kinetics experimentswere performed at 25° C. and 37° C. Kinetic dissociation constant(k_(d)) were determined by fitting the real-time sensorgrams to a 1:1binding model using Scrubber 2.0c curve fitting software. Bindingdissociative half-lives (t½) were calculated from k_(d) as:

${t\mspace{14mu} {1/2}\left( \min \right)} = \frac{\ln (2)}{60*{kd}}$

Half-life ratios for human C5 binding to different anti-C5 monoclonalantibodies at 25° C. and 37° C. in two running buffers PBS-T, pH7.4 andPBS-T, pH6.0 are shown in Tables 11 and 12.

TABLE 11 Half-life ratios of selected anti-C5 antibodies for human C5 at25° C. t½ Ratio mAb Captured pH 7.4/pH 6.0 H4H12169P IC H4H12176P2 ICH4H12161P IC H4H12159P ≤0.3 H4H12170P ≤0.5 H4H12166P 4.5 H4H12183P2 ICH4H12167P 0.6 H4H12164P 0.3 H4H12163P 1.2 H4H12175P 0.9 H4H12177P2 ≤0.5H4H12171P 0.6 H4H12168P 1.5 H4H12166P2 9.3 H4H12166P3 7.9 H4H12166P4 7.8H4H12166P5 8.3 H4H12166P6 7.8 H4H12166P7 35 H4H12166P8 47 H4H12166P9 31H4H12166P10 33 H4H11683N 2 H4H11686N 2 IC = inconclusive

TABLE 12 Half-life ratios of selected anti-C5 antibodies on human C5 at37° C. t½ Ratio pH 7.4/ mAb Captured pH 6.0 H4H12169P IC H4H12176P2 ≤0.4H4H12161P ≤0.7 H4H12159P ≤0.2 H4H12170P ≤0.2 H4H12166P 3.8 H4H12183P2 ICH4H12167P 0.2 H4H12164P ≤0.1 H4H12163P 0.8 H4H12175P 0.9 H4H12177P2 1.3H4H12171P 3.7 H4H12168P 1 H4H12166P2 7.3 H4H12166P3 6.6 H4H12166P4 7.6H4H12166P5 7.6 H4H12166P6 8.2 H4H12166P7 21 H4H12166P8 36 H4H12166P9 28H4H12166P10 19 H4H11683N 1.4 H4H11686N 0.8 IC = inconclusive

Half-life ratios for monkey C5 binding to different anti-C5 monoclonalantibodies at 25° C. and 37° C. in two running buffers PBS-T, pH7.4 andPBS-T, pH6.0 are shown in Tables 13 and 14.

TABLE 13 Half-life ratios of selected anti-C5 antibodies on monkey C5 at25° C. t½ Ratio mAb pH 7.4/ Captured pH 6.0 H4H12169P 3.4 H4H12176P2≥9.1 H4H12161P IC H4H12159P 1.2 H4H12170P ≥1.7 H4H12166P 18.5 H4H12183P25.8 H4H12167P 9.2 H4H12164P 2.9 H4H12163P 9.7 H4H12175P 3.6 H4H12177P23.7 H4H12171P 2.1 H4H12168P 3.8 H4H11683N 0.34 H4H11686N 0.37 IC =inconclusive

TABLE 14 Half-life ratios of selected anti-C5 antibodies on monkey C5 at37° C. mAb t½ Ratio Captured pH 7.4/pH 6.0 H4H12169P 2 H4H12176P2 2.8H4H12161P 10.7 H4H12159P 6.3 H4H12170P 4.7 H4H12166P 7.1 H4H12183P2 2.4H4H12167P 4.4 H4H12164P 1.1 H4H12163P 3.3 H4H12175P 0.4 H4H12177P2 1.5H4H12171P 4.7 H4H12168P 4 H4H11683N 0.7 H4H11686N 0.5 IC = inconclusive

As shown in Tables 11-14, selected anti-C5 antibodies showedpH-dependent binding, as seen by the t½ ratios.

Example 5: OCTET® Cross-Competition Between Anti-C5 Antibodies

Binding competition between anti-C5 monoclonal antibodies (mAbs) wasdetermined using a real time, label-free bio-layer interferometry assayon an OCTET® RED384 biosensor (Pall ForteBio Corp.). The entireexperiment was performed at 25° C. in 0.01 M HEPES pH7.4, 0.15M NaCl,0.05% v/v Surfactant Tween-20, 0.1 mg/mL BSA (OCTET® HBS-P buffer) withthe plate shaking at the speed of 1000 rpm. To assess whether 2antibodies were able to compete with one another for binding to theirrespective epitopes on a human C5 (hC5 purified from plasma, EMD),around 1.5 nm of anti-human C5 mAb was first captured onto anti-hFcantibody coated OCTET® biosensor tips (Pall ForteBio Corp., #18-5060) bysubmerging the tips for 3 minutes into wells containing a 50 μg/mLsolution of anti-human C5 mAb (subsequently referred to as mAb1). Theantibody captured biosensor tips were then saturated with a blocking H4Hisotype control mAb (subsequently referred to as blocking mAb) bydipping into wells containing 200 μg/mL solution of blocking mAb for 4minutes. The biosensor tips were then subsequently dipped into wellscontaining a co-complexed solution of 50 nM hC5 and 1 pM of a secondanti-human C5 mAb (subsequently referred to as mAb2), that had beenpreincubated for 2 hours, for 4 minutes. The biosensor tips were washedin OCTET® HBS-P buffer in between every step of the experiment. Thereal-time binding response was monitored during the course of theexperiment and the binding response at the end of every step wasrecorded. The response of human C5 pre-complexed mAb2 binding to mAb1was corrected for background binding, compared andcompetitive/non-competitive behavior of different anti-C5 monoclonalantibodies was determined.

Table 15 explicitly defines the relationships of antibodies competing inboth directions, independent of the order of binding.

TABLE 15 Cross-competition between pairs of selected anti-C5 antibodiesFirst mAb (mAb1) Captured using AHC mAb2 Antibodies Shown to Competewith Octet Biosensors mAb1 H4H12183P2 H4H12167P; H4H12166P; H4H12163PH4H12167P H4H12183P2; H4H12166P; H4H12163P H4H12166P H4H12183P2;H4H12167P; H4H12163P H4H12163P H4H12183P2; H4H12167P; H4H12166PH4H12159P H4H12169P; H4H11683N; H4H12170P H4H12169P H4H12159P;H4H11683N; H4H12170P H4H11683N H4H12159P; H4H12169P; H4H12170P H4H12170PH4H12159P; H4H12169P; H4H11683N H4H12175P H4H12177P2 H4H12177P2H4H12175P H4H12176P2 H4H12164P H4H12164P H4H12176P2 H4H12168P noneH4H12161P none H4H11686N none

Example 6: Inhibition of C5-Mediated Complement-Dependent Cytotoxicityin a B-Cell Bioassay

This Example describes a bioassay to test the role of C5 using ananti-CD20 antibody in the classical complement pathway. Therapeuticanti-CD20 antibodies against the B-cell specific cell-surface antigenCD20, have been shown to lead to CDC of B-cells (Glennie et al. 2007,Mol. Immunol. 44: 3823-3837) and CDC assay using cell lines expressingCD20 has been described previously (Flieger et al. 2000, Cell. Immunol.204: 55-63). Daudi cells, a human B cell line expressing CD20,complement preserved serum or C5 depleted serum with exogenous C5variants and an anti-CD20 antibody (antibody comprising VH/VL of “2F2”from U.S. Pat. No. 8,529,902) were used to assess the role of C5activity in CDC.

For the C5 CDC bioassay, Daudi cells were seeded onto a 96-well assayplates at 10,000 cells/well in either RPMI containing 10% FBS,penicillin/streptomycin, L-glutamine, sodium pyruvate and Non-EssentialAmino Acids (RPMI Complete media) or RPMI containing 1% BSA,penicillin/streptomycin and L-glutamine (RPMI/BSA). All assays testingmutated anti-hC5 antibodies, along with testing of the non-mutatedantibodies with C5 containing human serum were tested in RPMI Completemedia, while assays testing the non-mutated antibodies with AfricanGreen monkey serum and human C5 variants were tested in RPMI/BSA media.To measure CDC with human or monkey serum, the anti-CD20 antibody wasdiluted 1:3 from 100 nM to 2 pM (including a control sample containingno antibody) and incubated with cells for 10 minutes at 25° C. followedby addition of 1.66% serum or 1.66% of C5 depleted serum and 6.6 nM C5variant proteins. The amount of C5 protein to be added to the C5depleted serum was based on the reported value of C5 concentration inhuman serum of 0.37 uM (Rawal et al 2008, J. Biol. Chem. 283:7853-7863). To test C5 antibody inhibition of CDC, C5 antibodies werediluted 1:3 from 100 nM to 2 pM (including a control sample containingno antibody) and incubated with 1.66% serum or 1.66% of C5 depletedserum and 6.6 nM C5 variant proteins for 30 minutes. Ten minutes priorto addition of antibodies with serum to cells, the anti-CD20 antibodywas added to cells at 1 nM, 2 nM, 3 nM, 3.5 nM, 7 nM, 10 nM, or 30 nM.At the conclusion of the incubation with the anti-CD20 antibody, theantibody/serum mixture was added to cells. Cytotoxicity was measuredafter 3.5 hours of incubation at 37° C. and in 5% CO₂, followed by 15minute incubation at 25° C., and addition of CYTOTOX-GLO™ reagent(PROMEGA™, # G9292). CYTOTOX-GLO™ is a luminescence-based reagent thatmeasures cell killing such that increased luminescence is observed withincreased cytotoxicity (measured in relative light units, RLUs).Untreated cells in control wells were lysed by treatment with digitoninimmediately after addition of CYTOTOX-GLO™ reagent to determine maximalkilling of cells. Plates were read for luminescence by a VICTOR™ Xinstrument (Perkin Elmer) 15 minutes following the addition ofCYTOTOX-GLO™. Where calculated, the percentage of cytotoxicity wascalculated with the RLU values by using the following equation:

${\% \mspace{14mu} {Cytoxicity}} = {100 \times \frac{\left( {{{Ex}\; {perime}\; {ntal}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Back}\; {ground}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}{\left( {{{Maximum}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Backg}\; {round}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}}$

In this equation “background cell lysis” is the luminescence from thecells treated with media and serum alone without any anti-CD20 antibodyand the “maximum cell lysis” is the luminescence from the cells treatedwith digitonin. The results, expressed as % cytotoxicity or RLUs, wereanalyzed using nonlinear regression (4-parameter logistics) with PRISM™5 software (GRAPHPAD™) to obtain EC₅₀ and IC₅₀ values. Inhibition ofantibodies was calculated such that 0-100% inhibition is the range ofinhibition of the concentration of anti-CD20 antibody used in the assaywithout inhibitor to OnM anti-CD20 antibody.

Results

A total of 25 anti-human C5 antibodies, 16 non-mutated and 9 mutated,were tested for their ability to inhibit C5 in the CDC assay using Daudicells with an anti-CD20 antibody and either human sera (with normal hC5or C5 variants) or African green monkey sera. Various residues in thecomplementary determining regions (CDRs) of H4H12166P were mutated tohistidines to generate 9 mutated antibodies, H4H12166P2-H4H12166P10.Histidine mutations in the CDRs have been shown to confer pH-dependenceof binding to target antigen leading to improved pharmacokinetics (Igawaet al. 2010, Nat. Biotechnol.28: 1203-1207).

TABLE 16 Non-mutated anti-hC5 antibody inhibition of CDC with 1.66%serum and anti-CD20 antibody in Daudi Cells C5 depleted Human and C5depleted 6.6 nM C5 Human and African Green Variant 6.6 nM C5 Serum HumanHuman monkey R885H Variant R885C EC50 [M] of anti- 1.0E−09 1.4E−092.4E−09 1.9E−09 2.7E−09 CD20 antibody (with 1.66% Serum) Constantanti-CD20 1 nM 3 nM 3.5 nM 30 nM antibody (with 1.66% serum) IC50 [M](Max % IC50 [M] (Max Antibody IC50 [M] Inhibition)* IC50 [M] %Inhibition)* H4H11683N Not Tested 1.2E−09 4.0E−09 1.3E−09 9.0E−10H4H11686N Not Tested 1.5E−09 4.4E−09 1.1E−09 4.5E−10 H4H12159P 3.2E−09Not Tested 3.4E−09 1.4E−09 1.0E−09 H4H12161P 2.4E−09 Not Tested 2.6E−091.8E−09 1.0E−09 H4H12163P 3.4E−09 Not Tested 3.7E−09 2.1E−09 1.1E−09H4H12164P 2.4E−09 Not Tested 5.8E−09 1.8E−09 8.2E−10 H4H12166P 2.6E−09Not Tested 4.5E−09 1.3E−09 4.6E−10 H4H12167P 2.5E−09 Not Tested 3.5E−091.9E−09 1.0E−09 H4H12168P 1.5E−09 Not Tested 2.0E−09 2.3E−09 8.6E−10H4H12169P 1.7E−09 Not Tested 2.9E−09 1.3E−09 6.7E−10 H4H12170P 2.0E−09Not Tested 3.7E−09 4.8E−10 4.3E−10 H4H12171P 1.9E−09 Not Tested 3.3E−091.6E−09 6.5E−10 H4H12175P 2.2E−09 Not Tested 5.2E−09 4.2E−09 >2.0E−08(67%) H4H12176P2 2.7E−09 Not Tested 3.5E−09 2.1E−09 1.3E−09 H4H12177P22.2E−09 Not Tested 6.1E−09 2.4E−09 1.6E−09 H4H12183P2 1.7E−09 Not Tested1.4E−08 1.2E−09 4.5E−10 Comparator 1 2.3E−09 1.8E−09 >9.0E−08   Noinhibition No inhibition (49%) Control mAb 1 No Inhibition No InhibitionNot Tested Not Tested Not Tested Control mAb 2 Not Tested Not Tested NoInhibition No Inhibition No Inhibition *Unless otherwise noted, allinhibition is ~100%

As shown in Tables 16 and 17, all 25 anti-hC5 antibodies showed completeinhibition of CDC mediated by C5 present in 1.66% of human serum. TheIC50s of the non-mutated antibodies ranged from 1.2 to 3.4 nM. The IC50sof the mutated antibodies ranged from 3.0 nM to 12 nM. The parental,non-mutated antibody H4H12166P gave complete inhibition with IC50s of2.6 nM and 2.9n M.

TABLE 17 Mutated anti-hC5 antibody inhibition of CDC with 1.66% serumand anti-CD20 antibody in Daudi Cells C5 depleted C5 depleted Human andHuman and African Green 6.6 nM C5 6.6 nM C5 Serum Human monkey VariantR885H Variant R885C EC50 [M] of anti- 1.9E−09 2.6E−09 6.3E−09 9.5E−09CD20 antibody (with 1.66% Serum) Constant anti- 2 nM 10 nM 7 nM 30 nMCD20 antibody (with 1.66% serum) IC50 [M] (Max % Antibody IC50 [M]Inhibition)* IC50 [M] IC50 [M] H4H12166P 2.9E−09 5.6E−09    1.3E−097.6E−10 H4H12166P2 3.7E−09 9.7E−09    1.7E−09 1.2E−09 H4H12166P37.8E−09 >3.0E−08 (64%) 2.9E−09 1.7E−09 H4H12166P4 3.5E−09 7.9E−09   1.5E−09 9.6E−10 H4H12166P5 4.9E−09 >3.0E−08 (75%) 2.1E−09 1.4E−09H4H12166P6 3.0E−09 9.9E−09    1.3E−09 7.9E−10 H4H12166P77.3E−09 >6.0E−08 (61%) 4.2E−09 2.3E−09 H4H12166P8 4.1E−09 >2.0E−08 (79%)2.1E−09 1.2E−09 H4H12166P9 3.9E−09 >1.0E−08 (85%) 1.7E−09 7.7E−10H4H12166P10 1.2E−08 >1.0E−07 (34%) 7.0E−09 3.5E−09 Comparator 12.7E−09 >1.0E−07 (35%) No Inhibition No Inhibition Control mAb 2 NoInhibition No Inhibition No Inhibition No Inhibition *Unless otherwisenoted, all inhibition is ~100%

The sixteen non-mutated anti-hC5 antibodies showed complete inhibitionof CDC mediated by African Green monkey C5 with IC50s ranging from 2.0nM to 14 nM.

Four of the 9 mutated antibodies showed complete inhibition of CDCmediated by African Green monkey C5 with IC50s ranging from 7.1 nM to9.9 nM. The remaining six mutated antibodies were blockers with IC50sgreater than 10 nM, and maximum inhibition (at 100 nM antibody) rangingfrom 34% to 85%. The parental, non-mutated antibody H4H12166P gavecomplete inhibition with IC50s of 4.5 nM and 5.6 nM.

To test whether the anti-hC5 antibodies inhibit human C5 variants, R885Hand R885C, C5-Depleted Human Serum was tested with 6.6 nM of each C5variant. All 25 anti-hC5 antibodies showed complete inhibition of CDCmediated by C5 variant R885H, with IC50s of the non-mutated antibodiesranging from 0.48 nM to 4.2 nM, while IC50s of the mutated antibodiesranged from 1.3 nM to 7.0 nM. The parental, non-mutated antibodyH4H12166P gave complete inhibition with IC50s of 1.3 nM and 1.3 nM.

Fifteen out of 16 non-mutated anti-hC5 antibodies showed completeinhibition of CDC mediated by C5 variant R885C with IC50s ranging from0.43 nM to 1.6 nM. One non-mutated antibody showed weak inhibition ofCDC with maximum inhibition of 67% (at 100 nM antibody) and an IC50>20nM. All nine mutated antibodies showed complete inhibition of CDCmediated by C5 variant R885C with IC50s ranging from 0.77 nM to 3.5 nM.The parental, non-mutated antibody H4H12166P gave complete inhibitionwith an IC50s of 0.46 nM and 0.76 nM.

Anti-CD20 antibody showed CDC of Daudi cells with 1.66% serum with EC50sof 1.0 nM, 1.4 nM, and 1.9 nM for human serum, 2.4 nM and 2.6 nM forAfrican Green monkey serum, 1.9 nM and 6.3 nM for hC5 depleted serumwith hC5 variant R885H, and 2.7 nM and 9.5 nM for hC5 depleted serumwith hC5 variant R885C. Neither of the irrelevant IgG controlantibodies, Control mAb1 and Control mAb2, demonstrated any inhibitionof CDC.

Example 7: Inhibition of C5a Activity as Determined by Luciferase Assay

This Example describes an assay to test the activation of C5a throughone of its receptors, C5aR1. C5aR1 is a G-protein coupled receptor(GPCR) and can initiate various GPCR coupled signaling pathways (Monk etal. 2007, Br. J. Pharmacol. 152: 429-448). A bioassay was establishedusing HEK293 cells stably transfected with human C5aR1 (Accession No.NP_001727.1) and human Gα16 (Accession No. NP_002059.3) along with aluciferase reporter [NFAT response element (4×)-luciferase]. Gα16 is arelatively promiscuous G protein that can couple to different types ofGPCRs leading to PLC-β activation and subsequent elevation of Ca^(++,)which in turn activates NFAT translocation and reporter genetranscription (Kostenis et al. 2005, Trends Pharmacol. Sci. 26:595-602). The resulting cell line, HEK293/hGα16/hC5aR1/NFAT-luc, wasisolated and maintained in 10% DMEM containing 10% FBS, NEAA,pencillin/streptomycin, 500 μg/mL G418, 100 μg/mL hygromycin B, and 7μg/mL blasticidin.

For the C5a luciferase bioassay, HEK293/hGα16/hC5aR1/NFAT-luc cells wereseeded into 96-well assay plates at 20,000 cells/well in OPTI-MEM™reduced serum medium (Invitrogen, #31985-070) supplemented with 0.5%BSA, penicillin/streptomycin and L-glutamine, and then incubated at 37°C. and 5% CO₂ overnight. BSA was used instead of FBS, since serum hasbeen shown to cleave and inactivate hC5a (Klos et al., 2013, Pharmacol.Rev. 65: 500-543). The next morning, hC5a was titrated from 100 nM to 2pM (including a control sample containing no hC5a) and added to cells todetermine the dose response titration curve for the cell line. To testhC5a antibody inhibition of hC5a, 500 pM of hC5a was added to cells.Immediately afterwards, antibodies diluted 1:3 from 100 nM to 2 pM(including a control sample containing no antibody) were added to cells.Cells were incubated for 5.5 hours at 37° C. in the presence of 5% CO₂.The luciferase activity was detected after the incubation with ONEGLO™reagent (PROMEGA™, # E6051). ONEGLO™ is a luminescence-based reagentthat measures the amount of luciferase present in cells. In this assay,increased hC5a activation leads to increased luciferase production andluminescence (measured in relative light units, RLUs). Measurement ofluminescence was performed using a VICTOR™ X instrument (Perkin Elmer).The results were analyzed using nonlinear regression (4-parameterlogistics) with PRISM™ 5 software (GRAPHPAD™) to obtain EC₅₀ and IC₅₀values. Inhibition of antibodies was calculated such that 0-100%inhibition is the range of inhibition from 500 pM hC5a without inhibitorto OnM hC5a.

Four anti-hC5 antibodies were tested for their ability to inhibit hC5aactivation of its receptor, hC5aR1, by measuring the extent ofinhibition of 500 pM hC5a activation of HEK293/hGα16/hC5aR1/NFAT-luccells.

TABLE 18 Anti-hC5 antibody inhibition of 500 pM hC5a inHEK293/Gα16/hC5aR1/NFAT-luc cells EC₅₀[M] hC5a 3.9E−10 Inhibition of 500pM hC5a mAb PID or REGN # IC₅₀ [M] H2aM11682N 4.6E−10 H2aM11684N 3.5E−11H2aM11694N 1.4E−10 H2aM11695N 4.2E−11 Control mAb No Inhibition

As shown in Table 18, all four antibodies of the invention, showedcomplete inhibition of 500 pM hC5a with IC50s ranging from 0.035 nM to0.46 nM. An Irrelevant IgG control antibody, Control mAb3, did notdemonstrate any inhibition of hC5a. hC5a activatedHEK293/Gα16/hC5aR1/NFAT-luc cells with an EC50 of 0.39 nM.

Example 8: Hemolysis Bioassay

Classical pathway hemolysis assay (CH) and alternative pathway hemolysisassay (AH) were developed to test the antibody activity.

The CH is a screening assay for the activation of the classicalcomplement pathway, which is sensitive to the decrease, absence, and/orinactivity of any component of the pathway. The CH tests the functionalcapability of serum complement components of the classical pathway tolyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep redblood cell antibody (hemolysin). When antibody-coated SRBC are incubatedwith test serum, the classical pathway of complement is activated andhemolysis results. If a complement component is absent, the CH levelwill be zero; if one or more components of the classical pathway aredecreased, the CH will be decreased. (Nilsson et al 1984, J. Immunol.Meth. 72: 49-59). This assay is used for characterization and screeningof high-affinity anti-human C5 antibodies.

Methods (A) Classical Pathway Complement Hemolysis Assay

Desired number of sheep red blood cells (SRBCs) were washed in GVB++buffer and re suspended at 1×10{circumflex over ( )}9 cells/mL. Tosensitize the SRBCs, were mixed with equal volume of the 1:50 dilutedrabbit anti-sheep hemolysin (1.5 mg/mL) at 37° C. for 20 minutes.Sensitized SRBC cells were diluted to 2×10{circumflex over ( )}8cells/ml in GVB++ prior to using in hemolysis assay. Normal human serumor cynomolgus monkey serum was diluted to 2% or 10% in GVB++ buffer. Totest the inhibition of C5 mediated hemolysis activity, test antibodieswere pre-incubated for 20 minutes at 4° C., at concentrations rangingfrom 0.6 nM to 800 nM in 2%-10% normal human or 10% cynomolgus monkeyserum or African green monkey serum. Round bottom 96 well plates wereused to measure hemolysis activity. A total of 100 ul sensitized sheepRBCs (2×10{circumflex over ( )}8 cells/ml) were plated into 96-wellplate followed by addition of 100 ul of respective serum samples thatwas pre-incubated with the test antibodies. Cells were gently mixed andincubated at 37° C. for 60 minutes. After the incubation time, cellswere spun down by centrifugation at 1250×g at 4° C. A total of 100 uL ofthe supernatant was transferred to a fresh 96 flat bottom plate and readat 412 nm on Spectramax microplate reader. The hemolytic activity wascalculated at final serum concentration of 1-5% for treatments.

Percent hemolysis was calculated as follows:

${\% \mspace{14mu} {Hemolysis}} = {100 \times \frac{\left( {{{Ex}\; {perime}\; {ntal}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Back}\; {ground}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}{\left( {{{Maximum}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Backg}\; {round}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}}$

In this equation “background cell lysis” is the OD at A412 nm from thecells incubated in GVB++ buffer only containing no serum. The “maximumcell lysis” is the OD at A412 nm from the cells treated with water. Theresults, expressed as % hemolysis were analyzed using nonlinearregression (4-parameter logistics) with PRISM™ 5 software (GRAPHPAD™) toobtain IC₅₀ values. Data represented as mean±Standard error of mean

(B) Alternative Complement Assay

Desired number of rabbit red blood cells (RbRBCs) were washed inGVB-Mg²⁺/EGTA buffer and re suspended at 2×10{circumflex over ( )}8cells/ml. Normal human or cynomolgus monkey serum was diluted to 10% inGVB-Mg²⁺/EGTA buffer. To test the inhibition of C5 mediated hemolysisactivity, antibodies at concentrations ranging from 3 nM to 800 nM werepre-incubated for 20 minutes at 4° C. in 5-10% normal human serum orcynomolgus monkey serum. Round bottom 96 well plates were used tomeasure hemolysis activity. A total of 100 ul RbRBCs (2×10{circumflexover ( )}8 cells/ml) were plated into 96-well plate followed by additionof 100 ul of 10% normal human serum or cynomolgus monkey serum orAfrican green monkey serum that was pre-incubated with the anti-C5antibodies. Cells were gently mixed and incubated at 37° C. for 60minutes. After incubation time, the cells were spun down bycentrifugation at 1250×g at 4° C. A total of 100 uL of the supernatantwas transferred to a fresh 96 flat bottom plate and read at 412 nm onSpectramax microplate reader. The hemolytic activity was calculated atfinal serum concentration of 5% serum.

Percent hemolysis was calculated as follows:

${\% \mspace{14mu} {Hemolysis}} = {100 \times \frac{\left( {{{Ex}\; {perime}\; {ntal}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Back}\; {ground}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}{\left( {{{Maximum}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Backg}\; {round}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}}$

In this equation “background cell lysis” is the OD at A412 nm from thecells incubated in GVB-Mg/EGTA buffer only containing no serum orwithout any anti-C5 antibody. The “maximum cell lysis” is the OD at A412nm from the cells treated with water. Inhibition by anti-C5 antibodies,IC₅₀ values were calculated using nonlinear regression (4-parameterlogistics) with PRISM™ 6 software (GRAPHPAD™)

Results (A) Inhibition of Human C5 Hemolysis

A total of 25 anti-human C5 (hC5) antibodies, 16 non-mutated and 9mutated, were tested for their ability to inhibit C5 from normal humanserum (NHS) in the CH50 assay using sensitized sheep red blood (SRBCs)and AH50 assay using rabbit red blood cells (RRBCs).

TABLE 19 Anti-hC5 antibody inhibition of CP and AP activity in 1% or 5%normal human serum (NHS) Human CP Human AP Human CP Human AP % Max % MaxmAb PID IC₅₀ [M] IC₅₀ [M] Inhibition Inhibition H4H12183P2 5.88E−091.60E−07 99.9% 78.9% H4H12176P2 4.58E−09 1.65E−08 94.1% 69.9% H4H12168P3.33E−09 2.85E−08 97.5% 66.2% H4H11686N 3.09E−09 1.30E−08 97.4% 76.2%H4H12167P 3.68E−09 1.55E−08 99.9% 64.8% H4H12161P 2.56E−09 2.55E−0893.7% 56.1% H4H12163P 2.72E−09 2.05E−08 96.1% 66.0% H4H12166P 2.80E−092.60E−08 95.0% 70.9% H4H11683N 2.54E−09 3.40E−08 98.1% 73.2% H4H12159P2.50E−09 1.75E−08 97.9% 73.4% H4H12177P2 2.34E−09 1.70E−08 97.5% 71.0%H4H12170P 2.39E−09 1.80E−08 98.2% 81.1% H4H12175P 2.36E−09 2.00E−0898.0% 80.2% H4H12171P 2.33E−09 1.55E−08 94.9% 42.0% H4H12164P 2.10E−091.45E−08 95.9% 69.7% H4H12169P 2.36E−09 2.00E−08 98.3% 44.5% Isotypecontrol No Activity No Activity No Activity No Activity

As shown in Table 19, sixteen anti-hC5 antibodies of this inventionshowed more than 94% inhibition of hemolysis in classical pathway (CP)mediated by C5 present in 1% of human serum. The IC50s of antibodiesranged from 2.1 to 5.9 nM and the percent inhibition ranged from95%-99%. All 16 anti-C5 antibodies showed more than 60% inhibition(except H4H12169P) in the alternative pathway (AP) hemolysis assaymediated by C5 present in 5% NHS. The IC50s of antibodies ranged from 13to 160 nM and the percent inhibition activity ranged from 44% to 81%.

TABLE 19 Anti-hC5 antibody inhibition of CP and AP activity in 1% or 5%normal human serum (NHS) Human CP Human AP Human CP Human AP % Max % MaxmAb PID IC₅₀ [M] IC₅₀ [M] Inhibition Inhibition H4H12166P 1.09E−082.09E−08 99.4% 86.9% H4H12166P2 1.59E−08 4.78E−08 98.2% 81.3% H4H12166P31.34E−08 6.00E−08 95.9% 78.3% H4H12166P4 1.32E−08 3.17E−08 98.6% 77.0%H4H12166P5 1.49E−08 6.55E−08 97.1% 77.7% H4H12166P6 1.03E−08 2.84E−0898.1% 82.4% H4H12166P7 2.43E−08 1.56E−07 93.7% 83.2% H4H12166P8 1.41E−087.30E−08 95.7% 73.3% H4H12166P9 1.16E−08 5.35E−08 93.7% 72.2%H4H12166P10 4.44E−08 No Activity 74.0% No Activity Isotype control NoActivity No Activity No Activity No Activity

As shown in Table 20, all 9 mutated anti-hC5 antibodies showedinhibition of CP and AP hemolysis activity mediated by C5 present in 5%of human serum. In the CP hemolysis assay, the parental, non-mutatedantibody H4H12166P showed more than 98% inhibition with IC50 of 10.9 nM.Eight mutated anti-hC5 antibodies showed more than 90% inhibition withIC50 ranging from 10.3 nM to 24.3 nM. Mutant anti-C5 antibody 12166P10showed partial inhibition of 74%. In the AP hemolysis assay theparental, non-mutated antibody H4H12166P showed more than 85% inhibitionwith IC50s of 20.9 nM. The mutated anti-hC5 antibodies showed inhibitionrange from 72-83% and the IC50s antibodies ranged from 28 nM to 0.15 μM.

(B) Inhibition of Monkey C5 Hemolysis

A total of 25 anti-human C5 (hC5) antibodies, 16 non-mutated and 9mutated, were tested for their ability to inhibit C5 from Cynomolgusmonkey and African green monkey in the CH50 assay using sensitized sheepred blood (SRBCs) and AH50 assay using rabbit red blood cells (RRBCs).

TABLE 21 Anti-hC5 antibody inhibition of CP and AP activity in 5% normalAfrican green monkey (AGM) sera AGM AGM AGM CP AGM AP Serum CP Serum AP% Max % Max mAb PID IC₅₀ [M] IC₅₀ [M] Inhibition Inhibition H4H12183P2No Activity No Activity No Activity No Activity H4H12176P2 3.04E−084.77E−08 91.0% 83.2% H4H12168P 2.80E−08 2.25E−08 90.8% 88.2% H4H11686N4.82E−08 1.63E−07 49.3% 50.4% H4H12167P 6.95E−08 6.95E−08 90.5% 53.4%H4H12161P 3.19E−08 4.75E−08 78.9% 35.7% H4H12163P 6.90E−08 2.16E−0783.2% 58.5% H4H12166P 1.30E−07 2.33E−07 81.0% 44.2% H4H11683N 2.92E−084.08E−08 81.1% 88.1% H4H12159P 2.58E−08 2.70E−08 93.4% 93.5% H4H12177P21.80E−07 1.01E−07 80.6% 8.80% H4H12170P 2.54E−08 2.69E−08 94.9% 90.9%H4H12175P 1.18E−07 9.85E−08 84.5% 17.4% H4H12171P No Activity 2.33E−0817.8% 69.70% H4H12164P 2.47E−07 1.78E−07 85.8% 15.60% H4H12169P 3.44E−089.15E−08 43.3% 45.70%

As shown in Table 21, the anti-hC5 antibodies showed different levels ofinhibition of CP or AP hemolysis activity in 5% African green monkeysera. In the CP assay, two of the 16 anti-hC5 antibodies showed noinhibition of the hemolysis activity. Fourteen antibodies showedinhibition ranging from 43-94%, with IC50s ranging from 25 nM to 180 nM.In the AP hemolysis assay, thirteen of the 17 antibodies showedinhibition activity ranging from 17%-93% with IC50s ranging from 22.5 nMto 233 nM.

TABLE 22 Anti-hC5 antibody inhibition of CP and AP activity in 5% normalCynomolgus (Cyno) monkey sera Cyno Cyno Cyno CP Cyno AP Serum CP SerumAP % Max % Max mAb PID IC₅₀ [M] IC₅₀ [M] Inhibition InhibitionH4H12183P2 1.42E−07 2.96E−08 64.6% 100.0% H4H12171P 8.70E−09 7.20E−0992.9% 98.8% H4H12170P 8.20E−09 7.00E−09 99.0% 99.2% H4H12159P 7.75E−097.05E−09 99.3% 99.6% H4H12168P 1.03E−08 5.45E−09 99.0% 99.7% H4H11683N9.00E−09 7.15E−09 98.8% 98.9% H4H12176P2 1.47E−08 7.70E−09 97.5% 99.3%H4H12161P 2.79E−08 7.80E−09 100.0% 98.4% H4H12169P 1.99E−08 7.95E−0992.8% 96.30% H4H11686N 1.41E−08 9.00E−09 94.5% 98.7% H4H12163P 1.65E−081.02E−08 96.4% 98.5% H4H12167P 2.13E−08 7.60E−09 100.0% 98.30% H4H12175P1.09E−08 8.05E−09 96.7% 98.10% H4H12166P 2.01E−08 8.85E−09 94.2% 98.60%H4H12177P2 1.71E−08 8.80E−09 94.90% 98.10% H4H12164P 1.96E−08 9.10E−0994.7% 98.70% Isotype control No Activity No Activity No Activity NoActivity

As shown in Table 22, anti-hC5 antibodies (except H4H12183P2, whichshowed 64% CP inhibition) showed more than 90% inhibition of CP or APhemolysis assay in 5% of Cynomolgus monkey serum. In CP hemolysis assay,the IC50s of antibodies ranged from 7.15 nM to 142 nM. In AP hemolysisassay, the IC50s of antibodies ranged from 5.4 to 29.6 nM.

(C) Inhibition of Variant Human C5 Hemolysis

Selected anti-C5 antibodies were tested for their ability to inhibitvariant human C5 (see Example 3 herein) from C5-depleted human serum inthe CH50 assay. In C5-depleted human serum supplemented with exogenousC5 variant R885H, H4H12166P, and Comparator 2 blocked CP hemolysis withIC50 values of 6.0 nM and 4.4 nM, respectively, and IC80 values of 7.6nM and 5.5 nM, respectively. For variant R885C, H4H12166P and Comparator2 blocked CP hemolysis in C5-depleted human serum with exogenous C5variants with an IC50 of 9.3 nM and 6.8 nM, respectively, and IC80values of 11 nM and 8.2 nM, respectively. As expected, Comparator 1 didnot block the hemolytic activity of human C5 variants.

(D) Inhibition of Human C5b-6 Complex

Selected anti-C5 antibodies were tested for their ability to inhibithuman C5b-6 complex from C5-depleted human serum in CH50 assay.H4H12166P potently blocked CP hemolysis in C5-depleted human serumsupplemented with exogenous huC5b-6 complex with an IC50 of 3.8 nM andIC80 value of 5.8 nM. In contrast, Comparator 1 blocked C5b-6complex-mediated hemolysis with lower potency, with IC50 values of 5.0nM and IC80 value of 46 nM respectively. Comparator 1 inhibited only 70%total hemolysis at the highest concentrations tested. Comparator 2 didnot block human C5b-6 complex hemolytic activity.

Example 9: Anti-C5 Antibodies Block Generation of C5a in CP HemolysisAssay

To assess whether anti-C5 antibodies inhibit the generation of C5a,supernatants from the assay for classical pathway (CP) hemolysis wereanalyzed for C5a levels by ELISA.

C5a, generated as the result of C5 cleavage, is a protein fragment of 74amino acids. C5a is rapidly metabolized by serum carboxypeptidases to amore stable, less active, 73 amino acid form, C5a des-Arg, by removal ofthe C-terminal arginine. The quantitation of C5a des-Arg thereforeprovides a reliable measurement for monitoring the generation of C5a invivo and in vitro. The MicroVue C5a ELISA kit used here detects C5ades-Arg according to information provided by the manufacturer.Preliminary experiments (data not shown) indicate that the primary 74amino acid form of C5a is also detected. For the purpose of thisExample, both forms will be collectively referred to as “C5a”.

C5a protein levels were determined in supernatants from the CP hemolysisassay using complement-preserved normal human serum (NHS) pre-incubatedwith H4H12166P or isotype control antibody as described in Example 8.C5a protein levels were measured using the MicroVue C5a ELISA kitaccording to the manufacturer's instructions. Briefly, samples werediluted and incubated on plates pre-coated with capture antibody (mouseanti-C5a specific for a neo-epitope on human C5a). Human C5a proteinprovided by the manufacturer was used as a standard for calibration. C5ain the supernatants was detected by HRP-conjugated detection antibody(mouse monoclonal antibody to the C5a region of C5). The chromogenicHRP-substrate, 3,3′,5,5′-tetramethylbenzidine (TMB), was added to detectHRP activity. A solution of 1N hydrochloric acid was used to stop thereaction, and the optical density at 450 nm (OD450) was measured on aSPECTRAMAX™ plate reader. Data were analyzed using nonlinear regression(4-parameter logistics) in GRAPHPAD™ PRISM™. C5a concentration wasexpressed as ng/mL of supernatant.

In the assay using 5% NHS, H4H12166P potently blocked increases in C5aprotein levels in a dose-dependent manner with an IC50 of 8.5 nM, whilethe isotype control antibody had no effect on C5a levels (FIG. 1).Maximal blockade at the highest tested H4H12166P concentration (267 nM)resulted in a ˜10-fold decrease in C5a levels to 3.8 ng/mL (0.3 nM),compared with 34 ng/mL (2.8 nM) observed at the lowest tested H4H12166Pconcentration (1 nM) in 5% serum. The C5a concentration observed formaximal blockade was close to the baseline C5a level of 2.3 ng/mL (0.2nM) in untreated 5% NHS.

Example 10: Characterization of Pharmacokinetics and Pharmacodynamics ofAnti-C5 Antibodies in Cynomolgus Monkey

This Example describes the characterization of the pharmacokinetics (PK)and pharmacodynamics (PD) of selected anti-C5 antibodies conducted inmale cynomolgus monkeys. Endogenous C5 levels were determined prior toanti-C5 antibody dosing and used to stratify animal dose groups.

Total circulating C5 levels in cynomolgus monkeys were determined usinga Human Complement C5 ELISA (Abcam, cat # ab125963), which was performedaccording to the manufacturer's recommendations. Average concentrationsof C5 protein in monkeys were determined to be 90.85 μg/mL±19.17 μg/mL.

For each anti-C5 antibody, 4 cynomolgus monkeys were each administered asingle intravenous (IV) injection at a dose of 15 mg/kg. Blood sampleswere collected from each animal from pre-dose through 1680 hours (70days), processed into serum and frozen at −80° C. until analyzed for PKand PD.

Total IqG Antibody Level Analysis by ELISA Immunoassay

Total antibody concentrations in monkey serum samples were measuredusing a non-validated direct ELISA. The ELISA procedure employed amicrotiter plate coated with a mouse anti-human IgG1/IgG4 Fc monoclonalantibody. Different anti-C5 antibodies were added to the plate and theanti-C5 antibodies captured on the plate were detected using abiotinylated mouse anti-human IgG4 Fc monoclonal antibody, followed byNeutrAvidin conjugated with Horseradish Peroxidase (NeutrAvidin HRP). Aluminol-based substrate specific for peroxidase was then added toachieve a signal intensity that is proportional to the concentration oftotal captured anti-C5 antibody. The relative light unit (RLU)measurements of the calibration standards and their respective nominalconcentrations were fitted using a weighted 4 Parameter Logisticequation to generate a calibration equation that described theconcentration of anti-C5 antibodies and response relationship of theassay. The lower limit of quantitation (LLOQ) was 1.56 ng/mL in theassay (2% monkey serum) and 78 ng/mL in neat monkey serum.

Determination of PK Parameters

PK parameters were determined by non-compartmental analysis (NCA) usingPhoenix®WinNonlin® software (Version 6.4, Certara, L.P.) and an IV bolusdosing model.

All PK parameters were derived from the respective mean concentrationvalues, including the observed maximum concentration in serum (C_(max)),the time of observed peak concentration, t_(max), and the estimatedhalf-life observed (T_(1/2)). For each antibody, the area under theconcentration versus time curve up to the last measureable concentration(AUC_(last)) and extrapolated from time zero to infinity (AUC_(inf))were determined using a linear trapezoidal rule with linearinterpolation and uniform weighting.

PD Analysis by Ex Vivo Hemolysis Assay

Pharmacodynamics of selected anti-C5 antibodies was analyzed using exvivo classical pathway and alternative pathway hemolysis assays.

Classical Pathway Hemolysis assay: Sheep red blood cells (SRBCs) werewashed in GVB++ buffer (Gelatin Veronal Buffer with CaCl2) and MgCl2)(Boston BioProducts) and re-suspended at 1×10{circumflex over ( )}9cells/mL. To sensitize the SRBCs, a total of 1×10{circumflex over( )}9/mL of SRBCs were mixed with equal volume of the 1:50 dilutedrabbit anti-sheep hemolysin (1.5 mg/mL) at 37° C. for 20 minutes.Sensitized SRBCs were diluted to 2×10{circumflex over ( )}8 cells/mL inGVB++ buffer prior to using in the hemolysis assay. Blood fromcynomolgus monkeys was collected prior to dosing and at 5 minutes, 4 and8 hours, and 1, 2, 3, 5, 7, 10, 14, 18, 21, 28, 35, 42, 49, 56, 63 and70 days post dose for PD analysis. Serum was prepared and frozen untilfurther use. On day of the assay, cynomolgus serum from respective timepoints was diluted to 10% in GVB++ buffer. Round bottom 96 well plateswere used to measure hemolysis activity. A total of 100 μl of sensitizedSRBCs (2×10{circumflex over ( )}8 cells/mL) were plated into 96-wellplate at 37° C. followed by addition of 100 ul of 10% cynomolgus monkeyserum from respective time points. SRBCs were gently mixed and incubatedat 37° C. for 10 minutes. After incubation time, the cells werecentrifuged at 1250×g at 4° C. A total of 100 μL of the supernatant wastransferred to a fresh 96 flat bottom plate and read at 412 nm on aSpectramax microplate reader. The hemolytic activity was calculated atfinal serum concentration of 5%. The percent hemolysis was calculatedwith the absorbance values by using the following equation:

${\% \mspace{14mu} {Hemolysis}} = {100 \times \frac{\left( {{{Ex}\; {perime}\; {ntal}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Back}\; {ground}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}{\left( {{{Maximum}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Backg}\; {round}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}}$

In this equation “background cell lysis” is the OD at A412 nm from theSRBCs incubated in GVB++ buffer only containing no serum. The “maximumcell lysis” is the OD at A412 nm from SRBCs treated with water. Theresults, expressed as % hemolysis were analyzed using nonlinearregression (4-parameter logistics) with PRISM™ 5 software (GRAPHPAD™) toobtain IC₅₀ values. Data are represented as mean±Standard Error of Mean.

Alternative Pathway Hemolysis assay: The desired number of rabbit redblood cells (RbRBCs) were washed in GVB-Mg²⁺/EGTA buffer and resuspended at 2×10{circumflex over ( )}8 cells/mL. Blood from cynomolgusmonkeys was collected prior to dosing and at 5 minutes, 4 and 8 hours,and 1, 2, 3, 5, 7, 10, 14, 18, 21, 28, 35, 42 and 49 days post dose forPD analysis. Serum was prepared and frozen until further use. Roundbottom 96 well plates were used to measure hemolysis activity. A totalof 100 μl RbRBCs (2×10{circumflex over ( )}8 cells/mL) were plated into96-well plate at 37° C. followed by addition of 100 μl of 10% cynomolgusmonkey serum from the respective time points listed above. RbRBCs weregently mixed and incubated at 37° C. for 60 minutes. After incubationtime, the cells were centrifuged at 1250×g at 4° C. A total of 100 μL ofthe supernatant was transferred to a fresh 96 flat bottom plate whichwas read at 412 nm on a Spectramax microplate reader. The hemolyticactivity was calculated for final serum concentration of 5% andexpressed as percentage of total hemolysis of RBCs by water. The percentof hemolysis was calculated as described above.

Results

Selected anti-C5 antibodies (listed in Table 1) were tested in initialexperiments for prolonged pharmacokinetic profiles in cynomolgus monkeysand C5-humanized mice (described in Example 10). H4H12166P and H4H12161Pwere selected as having high affinity coupled with prolonged PK and usedin subsequent experiments herein with Comparator 1 and Comparator 2.

Cynomolgus monkeys were administered a single 15 mg/kg IV bolus dose ofH4H12166P, H4H12161P, or Comparator 2. Serum concentrations of totalantibody and percent classical pathway (CP) hemolysis activity weredetermined at 19 time points during a 70-day in-life period. Alternativepathway (AP) hemolysis was determined at 17 time points during a 50-dayin-life period. Table 23 summarizes the mean antibody concentrations forall 3 antibodies. Mean total antibody concentrations versus timeprofiles are shown in FIG. 2. Mean PK parameters are described in Table24.

TABLE 23 Mean Concentrations of Total IgG in Serum Following a Single 15mg/kg Intravenous Injection of Selected Anti-C5 Antibodies to MaleCynomolgus Monkeys Time (hours Serum concentration of Ab (μg/mL) post-Mean ± SD dose) # H4H12166P H4H12161P Comparator 2 0 4 BLQ BLQ BLQ 0.0834 445 ± 30   456 ± 26.2 459 ± 55.2 4 4 328 ± 27   360 ± 28.2 363 ± 43.88 4 353 ± 29.9  316 ± 21.5 357 ± 16.1 24 4 282 ± 43.6  276 ± 32.4 248 ±19.6 48 4 225 ± 15.2  221 ± 21.5 212 ± 19.3 72 4 180 ± 15.0  181 ± 17.2196 ± 36.2 120 4 194 ± 20.9  162 ± 10.7 179 ± 23.3 168 4 171 ± 29.9  132± 17.0 157 ± 18.4 240 4 157 ± 12.7 96.1 ± 17.3 114 ± 13.0 336 4 120 ±10.2 49.3 ± 18.7 67.9 ± 25.8  432 4 105 ± 13.9 24.6 ± 11.8 42.6 ± 15.9 504 4 92.2 ± 10.6  13.8 ± 9.95 28.6 ± 13.1  672 4 75.1 ± 15.8  6.16 ±2.40 10.9 ± 6.25  840 3 59.6 ± 4.79  2.44 ± 0.85 4.45 ± 2.63  1008 343.3 ± 2.89  1.16 ± 0.52 2.17 ± 1.58  1176 3 30.6 ± 1.42  0.57 ± 0.251.29 ± 1.16  1344 3 25.9 ± 3.74  0.315 ± 0.16  0.492 ± 0.49  1512 3 18.2± 2.41  0.17 ± 0.08 0.270 ± 0.27  1680 3 11.5 ± 1.51  0.079 ± 0.07 0.123 ± 0.15  Time = Time in hours post single-dose injection; SD =Standard deviation; BLQ = Below Limit of Quantitation

Following IV bolus administration, the total IgG concentration-timeprofiles of H4H12166P, H4H12161P, and Comparator 2 were characterized byan initial brief distribution phase followed by single eliminationphases throughout the in-life period. Peak H4H12166P, H4H12161P, andComparator 2 concentrations were highly comparable, as correspondingC_(max)/Dose values between all the antibodies were within 1.1-fold(29.7, 30.4, and 30.6 [(ug/mL)/(mg/kg)], respectively) (Table 24).

TABLE 24 Mean Pharmacokinetic Parameters of Total IgG Concentrations inSerum Following a Single 15 mg/kg Intravenous Injection of SelectedAnti-C5 Antibodies to Male Cynomolgus Monkeys H4H12166P H4H12161PComparator 2 15 mg/kg IV (n = 4) Parameter Mean SD Mean SD Mean SDC_(max) (μg/mL) 445 30.0 456 26.2 459 55.2 C_(max) /Dose (μg/mL)/(mg/kg)29.7 2.00 30.4 1.75 30.6 3.68 C₀ (μg/mL) 448 30.5 458 26.6 461 55.6t_(max) (hours) 0.083 0 0.083 0 0.083 0 AUC_(last) day · (μg/mL) 50801040 2350 357 2810 470 AUC_(last)/Dose 339 69.3 157 23.8 187 31.3 day ·(μg/mL)/(mg/kg) AUC_(inf) day · (μg/mL) 5550 671 2350 356 2810 470AUC_(inf)/Dose 370 44.7 157 23.7 188 31.4 day · (μg/mL)/(mg/kg) CL(mL/h/kg) 0.114 0.0143 0.270 0.0411 0.228 0.0425 V_(ss) (mL/kg) 60.44.85 44.0 4.34 45.3 3.06 t_(1/2) (day) 15.6 1.43 5.50 2.45 5.91 1.13 IV= Intravenous; n = Number of animals; C_(max) = Peak concentration; C₀ =Initial concentration determined by extrapolation; t_(max) = Time toC_(max); AUC = Area under the concentration-time curve; AUC_(last) = AUCcomputed from time zero to the time of the last positive concentration;AUC_(inf) = AUC from time zero extrapolated to infinity; CL = Total bodyclearance; V_(ss) = Volume of distribution at steady state; t_(1/2 =)Half-life; SD = Standard Deviation. Note: t_(max) is expressed innominal hours

Assessment of concentration-time profiles revealed that H4H12166Pdemonstrated the slowest elimination with terminal antibodyconcentrations ≥10 μg/mL through study day 71. Kinetics of H4H12161P andof Comparator 2 were similar; both demonstrated more rapid eliminationthan H4H12166P, with mAb concentrations ≥10 μg/mL through day 22 and 29,respectively.

Consequently, dose-normalized exposures (AUC_(last)/Dose) indicated thatH4H12166P had the highest exposure at 339 day*(μg/mL)/(mg/kg), whileH4H12161P and Comparator 2 had approximately 2-fold lower exposure, 157and 187 day*(μg/mL)/(mg/kg), respectively, than that of H4H12166P.

Antibody half-life (t_(1/2)) calculated during the elimination phaseranged from 5.5 to 15.6 days across the dose groups and also correlatedwith exposure, as H4H12166P had the correspondingly highest t₁₁₂ of 15.6days, while H4H12161P and Comparator 2 had t_(1/2) values of 5.5 and 5.9days, respectively.

The pharmacologic effects of the anti-C5 antibodies from cynomolgusmonkey serum samples were determined ex vivo by complement classicalpathway (CP) hemolysis of sensitized sheep red blood cells (SRBCs) andalternative pathway (AP) hemolysis of rabbit red blood cells (RbRBCs).The inhibition of hemolytic activity was calculated for a final serumconcentration of 5% and expressed as percentage of total hemolysis ofRBCs by water. Table 25 summarizes ex vivo activity of the 3 antibodiesas determined by mean percent hemolysis.

TABLE 25 Ex Vivo Classical and Alternative Pathway Percent HemolysisActivity of Selected anti-C5 Antibodies Time (hours Classical Pathway %Hemolysis in Alternative Pathway % Hemolysis in post- cynomolgus serum,10 min, Mean ± SEM cynomolgus serum, 60 min, Mean ± SEM dose) #H4H12166P H4H12161P Comparator 2 H4H12166P H4H12161P Comparator 2 0 491.34 ± 7.6  Not tested 84.36 ± 20.28 73.44 ± 17.26 64.90 ± 19.51 55.77± 10.82 0.083 4 3.5 ± 1.4 Not tested 6.6 ± 6.5 5.53 ± 1.98 5.90 ± 3.923.83 ± 3.93 4 4 2.35 ± 1.06 Not tested 3.16 ± 2.2  7.43 ± 2.54 6.53 ±2.7  5.30 ± 2.80 8 4 1.55 ± 0.21 Not tested 1.25 ± 0.21 3.53 ± 0.91 4.70± 2.77 1.98 ± 0.83 24 4  7.7 ± 6.08 Not tested 4.55 ± 2.05 13.40 ± 2.77 4.68 ± 1.89 4.65 ± 2.35 48 4 2.85 ± 2.19 Not tested 2.6 ± 0.7 5.53 ±2.40 7.68 ± 5.22 2.28 ± 0.67 72 4  0.9 ± 0.42 Not tested  1.3 ± 0.287.95 ± 3.36 5.95 ± 2.23 1.45 ± 0.33 120 4 1.75 ± 0.07 Not tested  1.3 ±0.14 16.38 ± 6.91  7.60 ± 1.94 1.68 ± 0.22 168 4  1.6 ± 1.13 Not tested 1.4 ± 0.56 21.28 ± 8.24  10.75 ± 2.27  2.15 ± 0.19 240 4   1 ± 0.14 Nottested  3.7 ± 2.83 19.18 ± 10.20 13.53 ± 7.17  14.20 ± 16.73 336 4 2.55± 2.05 Not tested 37.85 ± 5.3  21.10 ± 7.55  50.58 ± 12.91 65.60 ± 26.04432 4 1.35 ± 0.91 Not tested 105.25 ± 3.3   15.20 ± 10.86 59.75 ± 12.6554.55 ± 19.11 504 4 3.55 ± 2.05 Not tested 107.1 ± 4.38  33.15 ± 8.80 88.55 ± 24.63 85.63 ± 27.48 672 4  2.2 ± 0.56 Not tested  88.9 ± 23.0575.25 ± 18.30 88.55 ± 8.53  91.58 ± 18.55 840 3 3.075 ± 2.70  Not tested105.37 ± 53.4  46.65 ± 5.30  92.33 ± 5.16  91.85 ± 2.33  1008 3 15.5 ±26.6 Not tested 108.85 ± 2.35  58.60 ± 9.48  92.45 ± 6.27  92.30 ± 2.69 1176 3 58.33 ± 39.55 Not tested 113.85 ± 2.62  72.95 ± 5.87  104.90 ±3.5   101.90 ± 0.42  1344 3 71.55 ± 43.02 Not tested 110.3 ± 1.98  Nottested Not tested Not tested 1512 3 91.375 ± 29.7  Not tested 110.6 ±0.85  Not tested Not tested Not tested 1680 3 112.22 ± 4.06  Not tested112.15 ± 0.5   Not tested Not tested Not tested Time = Time in hourspost single-dose injection; SEM = Standard error of mean; BLQ = BelowLimit of Quantitation; NC = Not calculated

As shown in Table 25 and FIG. 2, PD effects were measured by complementCP (10 minute incubation) to Day 70. H4H12166P blocked more than 95% ofCP hemolytic activity until day 35. Activity returned to pre-studymaximum hemolysis levels by day 70. Comparator 2 blocked about 95% of CPhemolytic activity through day 10, and activity rapidly returned topre-study maximum hemolysis levels by day 18.

PD effects were also measured by complement AP pathway (60 minuteincubation) hemolysis assays to day 49. As shown in Table 25 and FIG. 3,H4H12166P blocked 80% of the total AP hemolytic activity until day 18and activity returned to pre-study maximum hemolysis level at day 50.H4H1216P and Comparator 2 blocked 90% of AP hemolytic activity throughday 7, and activity returned to pre-study maximum hemolysis levels byday 21.

Example 11: Characterization of PK/PD of Anti-C5 Antibodies inC5-Humanized Mice

In this set of experiments, the pharmacokinetics and pharmacodynamics ofselected anti-C5 antibodies were assessed in mice humanized to expresshuman C5 protein using Velocigene® technology (Valenzuela et al 2003,Nat. Biotechnol. 21: 652-659). Humanized mice were engineered to replaceexon 2 through exon 41 of murine C5 gene with exons 2-42 of human C5gene (disclosed in US Patent Application Publication 2015/0313194,herein incorporated in its entirety).

Total circulating human C5 levels were determined using a HumanComplement C5 ELISA (Abcam, cat # ab125963), which was performedaccording to manufacturer's recommendations.

Determination of Total Drug Level in Serum by ELISA

Circulating anti-C5 antibody concentrations, both C5-bound and -unbound,were determined by total human antibody analysis using ELISA. Briefly, agoat anti-human IgG polyclonal antibody at 1 μg/mL in PBS wasimmobilized on 96-well plates overnight; plates were washed to removeunbound IgG and then blocked with 5% BSA. Serial dilutions of anti-C5antibody containing serum samples (6 points) and the reference standards(12 points) of the respective antibodies were transferred to theanti-human IgG coated plates and incubated for one hour. The plate-boundanti-C5 antibodies were then detected using a goat anti-human IgGpolyclonal antibody conjugated with horseradish peroxidase. Plates weredeveloped with TMB substrate according to the manufacturer's recommendedprotocol and signals of optical density (OD) at 450 nm were recordedusing a Perkin Elmer VICTOR™ X4 Multimode Plate Reader. Anti-C5 antibodyconcentrations in serum were calculated based on the reference standardcalibration curve generated using GRAPHPAD™ PRISM™ software.

Determination of PK Parameters

PK parameters were determined by non-compartmental analysis (NCA) usingPhoenix®WinNonlin® software (Version 6.3, Certara, L.P.) and anextravascular dosing model. Using the respective mean concentrationvalues for each antibody, all PK parameters, including estimatedhalf-life observed (t_(1/2)), and area under the concentration versustime curve up to the last measureable concentration (AUC_(last)) weredetermined using a linear trapezoidal rule with linear interpolation anduniform weighting.

PD Analysis by Hemolysis Assay

Pharmacodynamics of selected anti-C5 antibodies was determined using aclassical pathway complement hemolysis assay. Sheep red blood cells(SRBCs) (Sheep blood in Alsevers solution) were washed in GVB++ buffer(Gelatin Veronal Buffer with CaCl₂) and MgCl2) (Boston BioProducts) andre suspended at 1×10{circumflex over ( )}9 cells/mL. To sensitize,1×10{circumflex over ( )}9/mL of SRBCs were mixed with equal volume ofthe 1:50 diluted rabbit anti-sheep hemolysin (1.5 mg/mL) at 37° C. for20 minutes. Sensitized SRBCs were diluted to 2×10{circumflex over ( )}8cells/mL in GVB++ prior to use in the hemolysis assay. Serum samplesfrom pre-dosed animals or humanized C5 mice dosed with anti-C5antibodies collected on days 10, 20, 30, 40 and 50 post-dose werediluted to 20% in GVB++ buffer. A total of 100 μl sensitized SRBCs(2×10{circumflex over ( )}8 cells/mL) were plated into 96-well roundbottom plates at 37° C. followed by addition of 100 μl of 20% serum thatwas supplemented with 160-180 μg/mL human complement 3 (huC3) protein.Cells were gently mixed and incubated at 37° C. for 1 hour. Afterincubation, the cells were centrifuged at 1250×g at 4° C. A total of 100μL of supernatant was transferred to a fresh 96 flat bottom plate andread at A412 nm on a Spectramax microplate reader. The percent hemolysiswas calculated with the absorbance values by using the followingequation:

${\% \mspace{14mu} {Hemolysis}} = {100 \times \frac{\left( {{{Ex}\; {perime}\; {ntal}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Back}\; {ground}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}{\left( {{{Maximum}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Backg}\; {round}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}}$

In this equation “background cell lysis” is the OD at A412 nm from SRBCsincubated in GVB++ buffer only containing no serum. The “maximum celllysis” is the OD at A412 nm from SRBCs treated with water. The results,expressed as % hemolysis, were analyzed using nonlinear regression(4-parameter logistics) with PRISM™ 6 software (GRAPHPAD™) to obtainIC₅₀ values. Data represented as Mean±(Standard Error of Mean).

Experiment 1

In this experiment, the pharmacokinetics and pharmacodynamics ofexemplary antibody H4H12166P were assessed in comparison with Comparator1 and Comparator 2 in humanized C5 mice. Total circulating human C5levels were determined using a Human Complement C5 ELISA (Abcam, cat #ab125963), which was performed according to manufacturer'srecommendations. Average concentrations of human C5 in the mice weredetermined to be 39.73 μg/mL±17.82 μg/mL. There was a difference betweenmale (55.4±1.7 μg/ml, n=47) and female (24.7±0.6 μg/ml, n=49) mice.

Prior to antibody dosing, male and female humanized C5 mice werestratified according to human C5 levels that averaged 40 μg/mL. For eachanti-C5 antibody, cohorts of twenty-two mice received a single 15 mg/kgdose of H4H12166P, Comparator 1 or Comparator 2 by subcutaneous (s.c.)injection. All mice were bled predose and at one day post-injection forPK analysis. In addition, at 10, 20, 30, 40 and 50 days post injection,groups of 4 or 5 mice from each cohort were euthanized and terminalbleeds were collected for PK and PD analysis. Day 1 serum samples werethe mean of the entire cohort of 22 mice. Blood was processed into serumand frozen at −80° C. until analyzed.

Total antibody concentrations were determined at 7 time points andpercent hemolysis activity was determined at 6 time points over the50-day in-life period. Total anti-C5 antibody concentrations aresummarized in Table 26. The mean total antibody concentrations versustime profile are shown in FIG. 4. Mean PK parameters are described inTable 27.

TABLE 26 Mean Concentrations of Total IgG in Serum Following a Single 15mg/kg Subcutaneous Injection of anti-C5 antibodies in Humanized C5 miceSerum concentration of Ab (μg/mL) Time Mean ± SD (Day) # H4H12166PComparator 1 Comparator 2 1 22   178 ± 22.7  229 ± 40.7  164 ± 24.1 10 483.7 ± 22.2  102 ± 22.9  44 ± 24.1 20 5 57.1 ± 26.8  29 ± 31.3 11.4 ±10.3 30 5 38.1 ± 7.6  30.1 ± 34.2 3.6 ± 3.2 40 4 11.9 ± 5.0  0.4 ± 0.40.5 ± 0.4 50  4*  9.3 ± 12.2 0.3 ± 0.3 0.3 ± 0.2 Time = Time in hourspost single-dose injection; Day = Day of study; SD = Standard deviation;SEM = Standard error of mean; ND = Not detected; NS = No sample. *ForComparator 2, day 50, n = three due to the inability of one sample to beanalyzed due to technical issues.

TABLE 27 PK parameters Parameter Units H4H12166P Comparator 1 Comparator2 Day 1 mAb μg/mL 178 229 164 concentration AUC_(last) day · μg/mL 28012708 1418 t_(1/2) d 11.3 4.7 7.6 C_(max) = Peak concentration; AUC =Area under the concentration-time curve; AUC_(last) = AUC computed fromtime zero to the time of the last positive concentration; T_(1/2) =Estimated half-life observed

Mean concentration versus time profiles at day 1 show that the threeantibodies, H4H12166P, Comparator 1 and Comparator 2 had comparableserum concentrations of 178, 229 and 164 μg/mL, respectively. Comparator1 had a similar elimination profile to H4H12166P up to day 30, but atdays 40 and 50 exhibited a rapid increase in clearance versus H4H12166P.At day 50, H4H12166P had an average antibody serum concentration ofapproximately 9 μg/mL, whereas Comparator 1 and Comparator 2 both had a30-fold lower average antibody serum concentration of 0.3 μg/mL.Comparator 2 exhibited the lowest exposure of the three antibodiestested, with an approximately 2-fold lower AUC_(last) (1408 day·μg/mL)as compared to H4H12166P (2801 day·μg/mL) and Comparator 1 (2708day·μg/mL).

The pharmacologic effects of anti-C5 antibodies H4H12166P, Comparator 1and Comparator 2 from humanized C5 mouse serum samples supplemented withhuman C3 were measured out to day 50 and were determined ex vivo bycomplement classical pathway (CP) hemolysis of sensitized SRBC. Meanpercent hemolysis for each anti-C5 antibody is summarized in Table 28and the mean percent hemolysis versus time profile is shown in FIG. 5.

TABLE 28 Ex Vivo Classical Pathway Percent Hemolysis Activity ofanti-human C5 antibodies Classical pathway % hemolysis in 10% mouse Timeserum, 60 min, Mean ± SEM (Day) # H4H12166P Comparator 1 Comparator 2 122  NS NS NS 10 4  12.6 ± 7.79 10.39 ± 2.88 12.06 ± 9.12  20 5 18.8 ±8.1  21.59 ± 17.53 65.08 ± 52.87 30 5 13.76 ± 10.9 78.98 ± 40.3 91.67 ±16.74 40 4 41.71 ± 40.7 101.09 ± 4.01  68.99 ± 42.47 50  4*  62.2 ± 56.6 88.99 ± 17.51 105.14 ± 4.07  Time = Time in hours post single-doseinjection; Day = Day of study; SEM = Standard error of mean; ND = Notdetected; NS = No sample. *For Comparator 2, day 50, n = three due tothe inability of one sample to be analyzed due to technical issues

H4H12166P, Comparator 1 and Comparator 2 inhibited the terminalcomplement hemolytic activity that appeared to correlate with antibodyexposures. H4H12166P blocked more than 85% of hemolytic activity untilday 30 with activity returning to predose baseline levels by day 50.Comparator 1 and Comparator 2 blocked about 80% hemolytic activity untilday 20 and day 10, respectively, with activity returning to baselinelevels by day 30 for both.

Experiment 2

In this experiment, the pharmacokinetics and pharmacodynamics of anti-C5antibodies H4H12166P, H4H12161P, Comparator 1, and an isotype controlwas assessed in humanized C5 mice (mice homozygous for human C5expression). Total circulating C5 levels were determined using a HumanComplement C5 ELISA (Abcam, cat # ab125963), which was performedaccording to the manufacturer's recommendations. Average concentrationsof human C5 in the mice were determined to be 48.98 μg/mL±15.1 μg/mL.

Prior to antibody dosing, humanized male and female C5 mice werestratified according to human C5 levels that averaged 50 μg/mL. For eachanti-C5 mAb, cohorts of five mice received a single 15 mg/kgsubcutaneous (s.c.) injection of H4H12166P, H4H12161P, Comparator 1 oran isotype control. All mice were bled predose, 6 hours, 1, 2, 3, 4, 7,10, 13, 21, 30 and 45 days post injection for PK analysis. In addition,on day 59, all mice from each cohort were euthanized and terminal bleedswere collected for PK and PD analysis. Blood was processed into serumand frozen at −80° C. until analyzed.

Total antibody concentrations were determined at 12 time points andpercent hemolysis activity was determined at 1 time point during a59-day in-life period. Total serum antibody concentrations for eachanti-C5 antibody are summarized in Table 29. Mean total antibodyconcentration versus time profiles are shown in FIG. 6. Mean PKparameters are described in Table 30.

TABLE 29 Mean Concentrations of Total IgG in Serum Following a Single 15mg/kg Subcutaneous Injection of selected anti-C5 antibodies in HumanizedC5 mice Serum concentration of Ab (μg/mL) Mean ± SD Time Isotype (Day)H4H12166P H4H12161P Comparator 1 Control 0 ND ND ND ND 0.25 31.2 ± 4.243.5 ± 16.3 59.2 ± 24.1  61.5 ± 29.4 1 149.9 ± 16.1 193.8 ± 24.1  179.0± 9.8  218.1 ± 17  2 160.8 ± 20   221 ± 26.5 166.6 ± 22.3  188.8 ± 25.83 166.2 ± 12.4  210 ± 31.2 159.2 ± 33.2  177.9 ± 26.2 7 158.6 ± 8.5 162.5 ± 34.8  136.1 ± 38.1  184.9 ± 33.9 10 123.5 ± 28.7 133.2 ± 20.2 107.2 ± 45.7  159.5 ± 28.8 13  93.7 ± 23.6 97.2 ± 24.6 70.6 ± 38  117.2± 24.1 21  60.4 ± 14.9 42.4 ± 30.3 29.5 ± 20.6  80.0 ± 17.5 30  37.8 ±10.8 15.3 ± 19.7 4.2 ± 3.5 42.1 ± 6.7 45 20.7 ± 5.2 3.5 ± 5.2 0.4 ± 0.3 16.5 ± 13.9 59  4.1 ± 1.9 0.6 ± 1.0 0.08 ± 0.04  4.6 ± 4.5 Time = Timein hours post single-dose injection; Day = Day of study; SD = Standarddeviation; SEM = Standard error of mean,; ND = Not detected; NS = Nosample.

TABLE 30 PK parameters Test Antibody (mean ± SD) Parameter UnitsH4H12166P H4H12161P Comparator 1 Isotype control C_(max) μg/mL 178 ± 10225 ± 22 183 ± 18 221 ± 19 AUC_(last) d · μg/mL 3490 ± 590 3040 ± 9002240 ± 780 4080 ± 480 t_(1/2) D 11 ± 1 5.8 ± 2  4.2 ± 1  9.9 ± 4 C_(max) = Peak concentration; AUC = Area under the concentration-timecurve; AUC_(last) = AUC computed from time zero to the time of the lastpositive concentration; T_(1/2) = Estimated half-life observed.

Mean concentration versus time profiles show that H4H12166P, H4H12161P,Comparator 1 and isotype control reached a maximum serum concentration(C_(max)) between days 1 to 3, with comparable C_(max) values within1.3-fold (178, 225, 183 and 221) μg/mL, respectively. H4H12166P andisotype control had similar elimination profiles, with remaining druglevels of approximately 4 μg/mL at day 59. H4H12161P exhibited fasterclearance than H4H12166P and isotype control but cleared more slowlythan Comparator 1. At day 59, H4H12161P had mean serum drug level of 0.6μg/mL while Comparator 1 had an almost undetectable drug level of 0.08μg/mL.

The isotype control, H4H12166P and H4H12161P exhibited comparableexposure (AUC_(last)) values within 1.3-fold (4080, 3490 and 3040day·μg/mL, respectively) whereas Comparator 1 exhibited a 1.6-fold lowerexposure (2240 day·μg/mL) compared to H4H12166P.

Example 12: LC-MRM-MS-Based Assay to Determine the Concentration ofTotal Human C5

In this Example, serum concentrations of total human C5 were determinedusing a liquid chromatography coupled to multiple reaction monitoringmass spectrometry (LC-MRM-MS) method in apharmacokinetics/pharmacodynamics study of anti-C5 antibody H4H12166P.

The serum concentrations of total human C5 were determined by measuringthe concentration of a 10-amino acid peptide contained in the C5sequence LQGTLPVEAR (aa 1129-1138 of SEQ ID NO: 359) as a proxy for C5.Theoretically, this method could also detect the C5 split product, C5b.However, due to the instability of free C5b, concentrations C5b in serumare generally low with the majority of C5b being bound to cell surfacesin the form of MAC complexes (Cooper & Muller-Eberhard 1970, J. Exp.Med. 132: 775-93; Hadders et al 2012, Cell Rep. 1: 200-7). Therefore,the processed serum samples analyzed here are likely to contain onlynegligible amounts of C5b product, if any.

Methods

For the PK/PD study, mice received a single 15 mg/kg dose of H4H12166Pby subcutaneous (s.c.) injection. All mice were bled predose and at oneday post-injection for PK analysis. In addition, at 10, 20, 30, 40, 50and 60 days post injection, mice were euthanized and terminal bleedswere collected for PK and PD analysis.

Human C5 was used as a reference standard for calibration; and a humanC5 peptide produced with a C-terminal stable isotope-labeled arginineresidue was used as the internal standard (LQGTLPVEAR-¹³C₆ ¹⁵N₄).Reference standard was used at concentrations ranging from 3.9 to 250μg/mL (1:2 serial dilutions) in serum from in house-generated C5knock-out mice, in which the mouse C5 gene was deleted (C5−/−). Serumfrom C5−/− mice was also used as a negative control (blank). Calibrationstandards, blanks, and study serum samples (10 μL each) were dried andthen were denatured in 100 μL of 8M urea/20 mMTris(2-carboxyethyl)phosphine (TCEP) buffer at 37° C. for 1 hour. Next,10 μL of 25 nM internal standard was added to all samples. Samples werealkylated with 10 mM of 2-iodoacetamide at room temperature for 30minutes and were diluted using 50 mM ammonium bicarbonate to a finalvolume of 500 μL. The samples were then digested by trypsin (1:20 w/w)overnight at 37° C. The tryptic peptide LQGTLPVEAR derived from C5 wasdetected and quantified by LC-MRM-MS using a Waters Xevo TQ-S withACQUITY UPLC system. Each processed sample (10 μL) was injected onto apre-equilibrated ACQUITY UPLC BEH C18 Column. The flow rate was 0.6mL/min (Mobile Phase A:water:formic acid/100:0.1 [V:V] and Mobile PhaseB: acetonitrile:formic acid/100:0.1 [V:V]). Retention time and peak areawere determined using Masslynx Analyst Data software (Waters).Concentrations of C5 analyte were calculated from the calibration curvewhich was constructed by plotting the peak area ratio of C5 referencestandard (unlabeled C5 peptide LQGTLPVEAR-¹²C₆ ¹⁴N₄ generated by trypticdigest of hC5) to internal standard (stable isotope-labeled C5 peptide)versus the nominal concentration of C5 reference standard.Concentrations were calculated using linear regression. The lowestconcentration of C5 reference standard (3.9 μg/mL) was within thedynamic range of the assay and was defined as the assay's LLOQ.

Results

Concentrations of total human C5 in serum were evaluated for samplescollected and via tail bleed in advance of dosing (predose) and viaterminal bleed on days 10, 30 and 35, from the corresponding animals.Total hC5 concentrations following H4H12166P dosing were similar (within˜1 to 0.9-fold) to predose levels on days 10, 30, and 35 post dosing.The observed minor differences were not statistically significant asassessed by Mann-Whitney test using GRAPHPAD™ PRISM™ software. Analysisof the C5/H4H12166P molar ratio demonstrated that H4H12166P remained inmolar excess of C5 through day 35 post dosing (Table 31).

TABLE 31 Summary of PD characteristics of H4H12166P % CP C5 (μg/mL)H4H12166P Molar ratio Day Post Hemolysis % Fold (μg/mL) terminal doseMean ± SD Inhibition Predose Terminal Change mean ± SD C5:H4H12166P 077.2 0 n/d n/a n/a 10 5.2 93 34.2 30.0 1.0 78.8 0.3 30 7.4 90 31.9 28.40.9 30.4 0.8 35 6.0 92 36.1 32.7 1.0 29.6 0.9 40 37.1 52 n/d 20.9 n/d 50All animals in group excluded due to MAHA titers >1000 60 73.57 5 n/d5.9 n/d ^(a)Percentage of inhibition of CP hemolytic activity wascalculated from mean % CP hemolysis values on the indicated day postdosing relative to the mean % CP hemolysis value on day 0. ^(b)FoldChange = terminal (indicated day post dosing) C5: predose C5. SD =standard deviation; MAHA = mouse anti-human antibody; n/d = notdetermined Animals with MAHA-impacted data were completely excluded fromcalculations (2x day 30, 1x day 35, 2x day 40, and all 4 mice on day 50)

Example 13: Epitope Mapping of H4H12166P Binding to C5 byHydrogen/Deuterium Exchange

H/D exchange epitope mapping with mass spectrometry was carried out todetermine the amino acid residues of hC5 [(amino acids M1-C1676 of SEQID No: 359) with which H4H12166P interacts. A general description of theH/D exchange method is set forth in e.g., Ehring (1999) AnalyticalBiochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem.73:256A-265A.

HDX-MS experiments were performed on an integrated Waters HDX/MSplatform, consisting of a Leaptec HDX PAL system for the deuteriumlabeling, a Waters Acquity M-Class (Auxiliary solvent manager) for thesample digestion and loading, a Waters Acquity M-Class (pBinary solventmanager) for the analytical column gradient, and Synapt G2-Si massspectrometer for peptic peptide mass measurement.

The labeling solution was prepared in 10 mM PBS buffer in D₂O at pD 7.0(equivalent to pH 6.6). For deuterium labeling, 3.8 μL of C5 (6 pmol/μL)or C5 premixed with the antibody in 1:1 molar ratio was incubated with56.2 μL D₂O labeling solution for various time-points (e.g.,undeuterated control=0 sec, labeled for 1 min and 20 min). Thedeuteration was quenched by transferring 50 μL sample to 50 μLpre-chilled quench buffer (0.2 M TCEP, 6 M guanidine chloride in 100 mMphosphate buffer, pH 2.5) and the mixed sample was incubated at 1.0° C.for two minutes. The quenched sample was then injected into a Waters HDXManager for online pepsin/protease XIII digestion. The digested peptideswere trapped onto an ACQUITY UPLC BEH C18 1.7-μm, 2.1×5 mm VanGuardpre-column at 0° C. and eluted to an analytical column ACQUITY UPLC BEHC18 1.7-μm, 1.0×50 mm for a 9-minute gradient separation of 5%-40% B(mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formicacid in acetonitrile). The mass spectrometer was set at cone voltage of37 V, scan time of 0.5 s, and mass/charge range of 50-1700 Thomson units(Th).

For the identification of the peptides from human C5, LC-MS^(E) datafrom undeuterated sample were processed and searched against thedatabase including human C5, pepsin, and their randomized sequence viaWaters ProteinLynx Global Server (PLGS) software. The identifiedpeptides were imported to DynamX software and filtered by two criteria:(1) minimum products per amino acid=0.3 and (2) replication filethreshold=3. DynamX software then automatically determined deuteriumuptake of each peptide based on retention time and high mass accuracy(<10 ppm) across multiple time points with 3 replicates at each time.

Using the online pepsin/protease XIII column coupled with MS^(E) dataacquisition, total 189 peptides from human C5 were identified in theabsence or presence of the antibody, representing 62% sequence coverage.Five peptides had significantly reduced deuteration uptake (centroiddelta values >0.9 daltons with p-values <0.05) when bound to H4H12166Pand are illustrated in the Table 32.

TABLE 32 Deuteration of Human C5 peptides upon binding to H4H12166P 1min Deuteration 20 min Deuteration C5 C5 + H4H12166P C5 C5 + H4H12166PResidues Centroid H⁺ Centroid MH⁺ Δ Centroid MH⁺ Centroid MH⁺ Δ 591-5991015.38 ± 0.09 1014.44 ± 0.16 −0.93 1015.64 ± 0.04 1014.60 ± 0.08 −1.04593-599  769.41 ± 0.11  768.33 ± 0.05 −1.08  769.65 ± 0.01  768.30 ±0.004 −1.35 775-787 1693.81 ± 0.11 1692.85 ± 0.07 −0.96 1694.06 ± 0.041692.96 ± 0.02 −1.10 775-794 2439.62 ± 0.29 2438.42 ± 0.20 −1.20 2440.16± 0.06 2439.17 ± 0.21 −0.99 779-787 1141.14 ± 0.04 1140.21 ± 0.05 −0.931141.23 ± 0.03 1140.21 ± 0.02 −1.02

The recorded peptide mass corresponds to the average value of thecentroid MH⁺ mass from three replicates. These peptides, correspondingto amino acids 591-599 and 775-794, had slower deuteration rate uponbinding to H4H12166P. These identified residues also correspond to theresidues 591-599 and 775-794 of human C5 as defined by Uniprot entryP01031 (CO5_HUMAN; SEQ ID NO: 359).

Example 14: Effect of Anti-C5 Antibodies on Ocular Inflammation inExperimental Autoimmune Uveitis in Mice

The present study was undertaken to evaluate the role of C5 inexperimental autoimmune uveitis (EAU). Both genetic [C5 knockout (KO),C3/C5 double KO mice], and pharmacologic (anti-C5 antibody) experimentalapproaches were used.

Methods

Adult C57BL/6J mice (n=25, Jackson labs), C5 KO (n=13) and C3/C5 KO(n=8) mice (Regeneron Pharmaceuticals Inc.) were used. EAU was inducedby subcutaneous injection of human interphotoreceptor retinoid-bindingprotein peptide (IRBP, New England Peptide) in complete Freund'sadjuvant and intraperitoneal injection of pertussis toxin. Anti-mouse C5mAb or isotype control mAb was administered through subcutaneousinjections every 3 days from day 5 to 28. The anti-mouse C5 antibodyused in this study (M1M17628N) comprised a HCVR/LCVR of SEQ ID NOs:362/363. SPECTRALIS® HRA+OCT (Heidelberg Engineering, Inc.) was used toassess levels of inflammation on days −1, 7, 14, 21 and 28. All animalswere euthanized on day 28 for eye and blood collection. Hemolysis assaywith/without human C3 was performed to validate complement inhibition.Data were analyzed by ANOVA.

Results

Compared to wild type mice, inflammation occurrence (30-50%) andvitreous cell cluster counts were significantly decreased in C5 KO mice(p<0.01). Optical coherence tomography (OCT) scores in C5 KO mice alsosignificantly reduced 50% at week 3 (p<0.0001). Interestingly, in C3/C5double KO mice, there were significantly more vitreous cell clusters andhigher disease scores on day 28 compared to wild type mice (p<0.05). Inanimals that received anti-mC5 Ab (50 mg/kg), inflammation incidence andvitreous cell clusters were significantly lower compared to either notreatment or isotype control group on day 21 (p<0.01). At weeks 3 and 4,OCT scores in anti-C5 antibody-treated group were significantly lowercompared to no treatment or isotype control (p<0.0001). (FIG. 7)Hemolysis assays with/without human C3 confirmed the inhibition effectof anti-C5 antibody at week 4 (FIG. 8).

Conclusion

Ocular inflammation due to EAU was mitigated by inhibiting C5 activity,either by genetic deletion or pharmacologic inhibition with a specificanti-C5 antibody. C5 depletion delayed EAU occurrence and reduced OCTdisease score. These results indicate that C5 is a potential therapeutictarget for autoimmune uveitis. Anti-C5 antibody has protective effect onEAU disease in wild type mice. Our findings also suggest that C3 mightbe beneficial for EAU disease in mice.

Example 15: Effect of Anti-Human C5 Antibodies on ExperimentalAutoimmune Uveitis

This Example describes the effects of anti-C5 antibodies against humanC5 in a mouse model of experimental autoimmune uveitis (EAU). The miceused for this study were humanized to express human C5 protein usingVelocigene® technology (Valenzuela et al 2003, Nat. Biotechnol. 21:652-659). Humanized mice were engineered to replace exon 2 through exon41 of murine C5 gene with exons 2-42 of human C5 gene (disclosed in USPatent Application Publication 2015/0313194, herein incorporated in itsentirety).

Methods

Adult, male mice were immunized subcutaneously in each thigh with 150 μgof human interphotoreceptor retinoid-binding protein (IRBP) peptide 1-20(GPTHLFQPSLVLDMAKVLLD) (SEQ ID NO: 364) (Avichezer et al 2000, Invest.Ophthalmol. Vis. Sci. 41:127-131) in 0.2 ml emulsion of CFA,supplemented with Mycobacterium tuberculosis strain H37RA to 2.5 mg/ml.Mice were then inoculated intraperitoneally with 1.0 μg of pertussistoxin (PTX) to facilitate induction of cell-mediated antoimmunity bypromoting a Th1 polarization of the immune response (Thurau et al 1997,Clin. Exp. Immunol. 109: 370-376; Silver et al 1999, Invest. Ophthalmol.Vis. Sci. 40: 2898-2905). The animal body weights were monitored twice aweek.

Ophthalmic examinations were carried out on day −1, before EAU inductionand on days 7, 14, 21 and 28. Mice were anesthetized with ketamine (120mg/kg, IP) and xylazine (5 mg/kg, IP). Pupils were dilated using a 0.5%ophthalmic solution of Tropicamide, and the fundus of the eye wasexamined using a contact lens with a fundus camera in a SpectralisHeidelberg retinal angiography platform (HRA)+OCT system (HeidelbergEngineering, Carlsbad, Calif., USA).

A series of 61 lateral optical sections were obtained for each eye usingthe OCT function on the Spectralis HRA+OCT system (HeidelbergEngineering, Carlsbad, Calif., USA)). The OCT imaging area was centeredon the optic disc allowing for equal imaging above and below the opticnerve head. The retinal thickness was measured as the distance betweenthe bottom of the RPE layer to the inner limiting membrane of the eye.Measurements were taken 1500 μm from the optic disc, and the values from4 different retinal quadrants (e.g., superior, inferior, temporal andnasal) were averaged for a mean retinal thickness of the eye.

The severity of inflammatory cell infiltration into the vitreous alsowas graded in OCT images, by assessing the average number ofinflammatory cell clusters in the vitreous, in four lateral OCT scansthat transected the optic nerve, per eye.

An 4-point scale was developed for assessment of disease severity in OCTimages (OCT Scores) (Table 33).

TABLE 33 Scoring EAU in vivo using Optical Coherence Tomography (OCT)Grade Criteria 0 No Change 0.5 (Trace) Minor inflammatory cellinfiltration in the vitreous, primarily near the optic nerve head (<15clusters) 1 Minor inflammatory cell infiltration in the vitreous,primarily near the optic nerve head (<25 clusters); minor focalsubretinal lesions (grey spots) in the periphery; minor retinal folds inthe periphery; retinal vascular dilation; perivasculitis and vasculitis2 Moderate inflammatory cell infiltration in the vitreous more diffusebut not in the far periphery (<50 clusters); retinal layer disruptions;small- to medium- sized granuloma formations with retinal foldsprimarily in the periphery; vessel dilation; minor focal choroidalneovascularization; perivasculitis and vasculitis; minor retinal edema(<10 μm) 3 Moderate to severe diffuse inflammatory cell infiltration inthe vitreous (>50 clusters); diffuse retinal layer disruptions anddilated vessels in the inner nuclear layer; medium- to large- granulomaformations with retinal folds throughout the retina; severe retinalvascular dilation; perivasculitis and vasculitis; moderate diffusechoroidal neovascularization; moderate- to severe- retinal edema (10-40μm); minor retinal detachments 4 Severe diffuse inflammatory cellinfiltration in the vitreous (>70 clusters); diffuse layer disruptionsand dilated vessels in the inner nuclear layer; severe diffuse granulomaformation with retinal folds; severe diffuse choroidalneovascularization; perivasculitis and vasculitis; retinal degenerationor severe retinal edema (>20 μm loss or >40 μm gain respectively); largeretinal detachments

Statistical Analysis

Statistical analyses for parametric data (body weight, inflammatory cellclusters in the vitreous, and retinal thickness) were performed byone-way ANOVA test and Tukey's multiple comparison test. Fornonparametric data (OCT scores and histology scores) analyses wereperformed by the Kruskal-Wallis test and Dunn's test with the GRAPHPAD™PRISM™ version 5.0d software relative to isotype control or no-treatmentgroups. Data show mean values ±SEM. A p-value of less than 0.05 wasconsidered as statistically significant.

Results

In a first study (Study A), mice were treated subcutaneously every 3days from day 5 with isotype control antibody (50 mg/kg), or 10 mg/kg or50 mg/kg of H4H12170P. Treatment with 10 mg/kg H4H12170P resulted in areduction in inflammation and retinal damage (FIG. 9). Mice treated with10 mg/kg H4H12170P also showed a statistically significant reduction inOCT scores on day 21 and day 28 (FIG. 10).

In a second study (study B), mice were treated subcutaneously every 3days from day 6 with either isotype control (10 mg/kg), 3 mg/kg or 10mg/kg of H4H12166P, or with Comparator 2 (see Example 2 herein; “ControlConstructs used in the following Examples”). Treatment with H4H12166Peither at 3 mg/kg or 10 mg/kg produced a dose-related reduction in OCTscores that was statistically significant on days 14 to 28 (FIG. 11).Treatment with 10 mg/kg H4H12166P in C5 humanized mice starting 6 daysfollowing EAU induction resulted in a dose-related reduction ininflammation and retinal damage as determined by OCT obtained on day 14to 28 (FIG. 12).

For both studies, non-invasive, in-life evaluation by OCT showed aprogressive development of inflammation, increased retinal thickness andmorphological abnormalities in control animals following immunizationwith IRBP.

Conclusion

These experiments provide further pharmacological evidence that C5 playsa role in the pathogenesis of autoimmune uveitis. Pharmacologicdepletion of human C5 by fully human anti-human C5 antibodies postponedEAU incidence and reduced disease severity, establishing the efficacy ofthese antibodies in autoimmune uveitis.

Example 16: Effect of Anti-C5 Antibodies on Renal Ischemia-ReperfusionInjury

The present study was carried out to evaluate the role of C5 in renalischemia-reperfusion injury. Both genetic (using C3 knockout and C5knockout mice) and pharmacological approaches (using anti-C5 antibodies)were used. Ischemia-reperfusion model was induced by bilateral renalpedicle clamping for 45 min followed by 48 h of reperfusion. Shamlaparotomy served as controls. Anti-C5 antibody was administered at 50mg/kg intravenously as a single dose immediately after ischemia(curative); or subcutaneously as two doses, day −1 and day 1 surgery(preventive). The anti-C5 antibody used for this study was M1M17628Ncomprising HCVR/LCVR of SEQ ID NO: 362/363. Blood urea nitrogen (BUN)and serum creatinine markers were used to assess levels of disease andprotection in the mice.

TABLE 34 Percent change in blood urea nitrogen levels in mice treatedwith anti-C5 antibody (M1M17628N) in preventive and therapeutic modesRIRI + Veh RIRI + Iso. Ctl BUN, % Change Vs. Day 2 Day 2 RIRI +M1M17628N −37.19 −34.68 (Prev) RIRI + M1M17628N −53.70 −51.85 (Cur)

TABLE 35 Percent change in serum creatinine levels in mice treated withanti-C5 antibody (M1M17628N) in preventive and therapeutic modes RIRI +Veh RIRI + Iso. Ctl SCr, % Change Vs. Day 2 Day 2 RIRI + M1M17628N−53.09 −49.34 (Prev) RIRI + M1M17628N −59.40 −56.16 (Cur)

Compared to wild type mice, C3 and C5 knockout mice showed significantfunctional protection in the RIRI model of acute kidney injury, asevidenced by reduction in the blood urea nitrogen and serum creatininelevels. The anti-C5 antibody showed functional protection in RIRI modelin both preventive and therapeutic modes (Tables 34-35).

Example 17: Effect of Anti-C5 Antibodies on Lupus Nephritis

This Example describes the efficacy of anti-C5 antibodies in treatinglupus nephritis in a mouse model.

Systemic lupus erythematosus (SLE) is an autoimmune disorder caused byloss of tolerance to self-antigens, the production of autoantibodies anddeposition of complement-fixing immune complexes (ICs) in injuredtissues. SLE is characterized by a wide range of clinical manifestationsand targeted organs, with lupus nephritis being one of the most seriouscomplications. Complement activation in the kidneys of lupus nephritispatients contributes to inflammation and tissue damage. The efficacy ofanti-C5 antibodies in treating lupus nephritis was studied in NZBWF1mice, a spontaneous mouse model of lupus nephritis (Yang et al 1996,PNAS). The mice develop autoimmune disease resembling human SLE,autoantibodies to nuclear antigens and cell membrane proteins,hypergammaglobulinemia, albuminuria, proteinuria, initiate immunecomplex glomerulonephritis and die of kidney failure and end-stage renaldisease at 35 to 50 weeks of age.

For this study, 25-week old NZBWF1 mice were subcutaneously treated with30 mg/kg of isotype control, or anti-C5 antibodies twice a week for 8weeks followed by thrice a week for 10 weeks. The anti-mouse C5antibodies used for this study were M1M17628N and M1M17627N, comprisingHCVR/LCVR of SEQ ID NOs: 362/363 and 365/366, respectively.

Treatment with anti-C5 antibodies significantly improved survival ratein mice (FIG. 13). Both antibodies improved albuminuria at 8-14 weeks oftreatment (FIG. 14), and blood urea nitrogen levels at 12-16 weeks oftreatment (FIG. 15).

Example 18: Effect of Anti-C5 Antibodies Against Astrocyte Cell Death

Neuromyelitis optica (NMO) is an autoimmune disease of the centralnervous system (CNS) that mainly affects the optic nerve and spinalcord. In NMO, anti-aquaporin-4 autoantibodies (AQP4-Ab) cause damage toastrocytes by activating complement-dependent cytotoxicity (CDC). Thegoals of this study were to evaluate the role of the complement systemin NMO progression and the use of an antibody against a complementprotein as a potential therapeutic treatment for NMO.

Primary rat cortical astrocytes were obtained from cerebral brain cortexof post-natal rat pups and were cultured with AQP4-Ab (antibody “rAb-53”from US Patent Application Publication 2014/0170140; Bennett et al 2009,Ann. Neurol. 66: 617-629) and complement proteins to demonstratecell-mediated cytotoxicity. Then the experiments were repeated withaddition of an anti-C5 antibody to demonstrate blocking of the astrocytecell destruction.

To quantify cell death, a CYTOTOX-GLO™ luminescence cytotoxicity assaywas performed. The assay used various concentrations of anti-C5 antibody(0.001 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml, or1000 μg/ml) or an isotype control antibody.

In order to determine whether anti-C5 antibody could block the AQP4-Abinduced CDC, astrocytes were plated and the CYTOTOX-GLO™ CytotoxicityAssay was repeated to find an optimal dose of AQP4-Ab for that plating.The optimal concentration of AQP4-Ab was found to be 50 μg/mL, and in afollowing experiment a constant dose of AQP4-Ab (50 μg/mL) was used,while the dose of anti-C5 antibody was varied. As shown in FIG. 16, adecrease in RLU was seen (on average 300 k to on average 100 k) withincreasing amounts of anti-C5 antibody demonstrating that the anti-C5antibody blocked astrocyte cell death. For both experiments, the RLU didnot vary with the isotype control antibody. As shown in FIG. 16, anti-C5antibodies inhibited AQP4 Ab induced cytotoxicity on primary corticalastrocyte with IC50 of 15-17 nM.

In a subsequent study, anti-AQP4 antibody and anti-C5 antibody will beinjected into rat brains to assess the therapeutic efficacy againstcomplement-mediated cytotoxicity of astrocytes in CNS.

Example 19: Endothelial Assay

This Example describes an in vitro glomerular endothelial assay toexamine if anti-C5 antibodies block C5b-9 and C3 deposition.

Reproducible methods for evaluating inhibitory effects of drugcandidates on complement activation are essential for preclinicaldevelopment. Due to the complexity of complement activation pathways, anassay should use relevant cells and endpoints to the given therapeuticindication. Here, using an immortalized human glomerular endothelialcell line (HGECs), a complement C3 & C5 deposition model was validatedfor use evaluating the blocking activity of anti-C3 or C5 mAbs.

Methods

Human primary kidney glomerular endothelial cells (HGEC; Cell Biologics)were plated overnight in complete media into collagen I—coated blackclear bottom 96-well plates. The cells were treated with either PBS(control) or activated for 10 mins with 10 uM ADP. After PBS washing,50% human serum (complement-preserved, C3-depleted, or C5-depleted) wasadded for 4 hours. Anti-C5 antibodies were added at 1 mg/mL to the serumprior to the treatment. The cells were washed and fixed and probed withanti-C3b antibodies (Thermofisher) and/or anti-C5b-9 antibodies (Abcam),secondary antibodies and counterstained with DAPI. Images were capturedon ImagExpress and high content image analysis was used to quantifyfluorescent staining for each image and averaged per condition.

Results

C3 and C5b-9 deposition was observed on ADP-activated HGECs exposed tonormal human serum but not on non-activated HGECs (C3: 1.5×10⁷±1.0×10⁷;C5: 7.9×10⁶±6.6×10⁶, P<0.05 vs non-ADP-activated HGEC). The depositionof C3 and C5b-9 were significantly reduced on ADP-activated HGEC exposedto C3 or C5 depleted serum (C3: 3.3×10⁵±4.8×10⁴; C5: 1.5×10⁶±6.0×10⁵,P<0.05). Addition of a blocking anti-C5 mAb significantly reduced normalhuman serum derived C5b-9 deposition onto ADP-activated HGEC, depositionwas comparable to C5 depleted sera (C5 mAb: 1.02×10⁶±6.0×10⁵, ControlmAb 3.7×10⁶±1.6×10⁶, P<0.05 vs. control mAb).

CONCLUSION

These data demonstrate the utility of an in vitro human glomerularendothelial assay to model complement C3 & C5 deposition. In addition toin-vitro screening, this assay offers potential as a translational modelto evaluate anti-complement strategies in renal disease using patientderived serum samples.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

1-32. (canceled)
 33. A method of treating or ameliorating at least onesymptom or indication of a disease or disorder associated with C5 in ahuman subject in need thereof, the method comprising administering atherapeutically effective amount of a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and an antibody orantigen-binding fragment thereof that specifically binds C5 comprising aheavy chain variable region that comprises an HCDR1 that comprises anamino acid sequence as set forth in SEQ ID NO: 100, an HCDR2 thatcomprises an amino acid sequence as set forth in SEQ ID NO: 102, and anHCDR3 that comprises an amino acid sequence as set forth in SEQ ID NO:104; and a light chain variable region that comprises an LCDR1 thatcomprises an amino acid sequence as set forth in SEQ ID NO: 108, anLCDR2 that comprises an amino acid sequence as set forth in SEQ ID NO:110, and an LCDR3 that comprises an amino acid sequence as set forth inSEQ ID NO: 112; to the human subject.
 34. The method of claim 33,wherein the disease or disorder is selected from the group consisting ofatypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria,age-related macular degeneration, geographic atrophy, uveitis,neuromyelitis optica, multiple sclerosis, stroke, Guillain BarreSyndrome, traumatic brain injury, Parkinson's disease, a disorder ofinappropriate or undesirable complement activation, a hemodialysiscomplication, hyperacute allograft rejection, xenograft rejection,interleukin-2 induced toxicity during IL-2 therapy, an inflammatorydisorder, inflammation of an autoimmune disease, Crohn's disease, adultrespiratory distress syndrome, thermal injury, burns, frostbite, apost-ischemic reperfusion condition, myocardial infarction, capillaryleak syndrome, obesity, diabetes, Alzheimer's disease, schizophrenia,stroke, epilepsy, atherosclerosis, vasculitis, bullous pemphigoid, C3glomerulopathy, membraneproliferative glomerulonephritis, diabeticnephropathy, Alport's syndrome, progressive kidney failure, proteinurickidney disease, renal ischemia-reperfusion injury, lupus nephritis,post-pump syndrome in cardiopulmonary bypass, post-pump syndrome inrenal bypass, hemodialysis, renal ischemia, mesenteric arteryreperfusion after aortic reconstruction, infectious disease, sepsis, animmune complex disorder, an autoimmune disease, a renal disorder,rheumatoid arthritis, systemic lupus erythematosus, systemic lupuserythematosus nephritis, proliferative nephritis, hemolytic anemia,asthma, chronic obstructive pulmonary disease, emphysema, pulmonaryembolism, pulmonary infarct, pneumonia, and myasthenia gravis.
 35. Themethod of claim 33, wherein the disease or disorder is atypicalhemolytic uremic syndrome.
 36. The method of claim 33, wherein thedisease or disorder is paroxysmal nocturnal hemoglobinuria. 37.(canceled)
 38. The method of claim 33, wherein the pharmaceuticalcomposition is administered in combination with a second therapeuticagent.
 39. The method of claim 38, wherein the second therapeutic agentis selected from the group consisting of an anti-coagulant, ananti-inflammatory drug, an antihypertensive, an immunosuppressive agent,a lipid-lowering agent, an anti-CD20 agent, rituximab, an anti-TNFagent, infliximab, an anti-seizure agent, a C3 inhibitor, a secondanti-C5 antibody, and an anti-thrombotic agent.
 40. The method of claim33, wherein the pharmaceutical composition is administeredsubcutaneously, intravenously, intradermally, intraperitoneally, orally,intramuscularly or intracranially.
 41. The method of claim 33, whereinthe antibody or antigen-binding fragment thereof comprises a heavy chainvariable region that comprises the amino acid sequence set forth in SEQID NO:
 98. 42. The method of claim 33, wherein the antibody orantigen-binding fragment thereof comprises a light chain variable regionthat comprises the amino acid sequence set forth in SEQ ID NO:
 106. 43.The method of claim 33, wherein the antibody or antigen-binding fragmentthereof comprises a heavy chain variable region that comprises the aminoacid sequence set forth in SEQ ID NO: 98 and a light chain variableregion that comprises the amino acid sequence set forth in SEQ ID NO:106.
 44. The method of claim 33, wherein the antibody or antigen-bindingfragment thereof comprises a heavy chain that comprises an amino acidsequence of SEQ ID NO:
 353. 45. The method of claim 33, wherein theantibody or antigen-binding fragment thereof comprises a light chainthat comprises an amino acid sequence of SEQ ID NO:
 354. 46. The methodof claim 33, wherein the antibody or antigen-binding fragment thereofcomprises a heavy chain that comprises an amino acid sequence of SEQ IDNO: 353; and a light chain that comprises an amino acid sequence of SEQID NO:
 354. 47. The method of claim 46, wherein the antibody orantigen-binding fragment thereof is an antibody.
 48. The method of claim47, wherein the disease or disorder is paroxysmal nocturnalhemoglobinuria (PNH).
 49. The method of claim 47, wherein the disease ordisorder is myasthenia gravis.
 50. The method of claim 47, wherein thedisease or disorder is neuromyelitis optica.
 51. The method of claim 48,wherein the antibody is administered to the subject intravenously andsubcutaneously.
 52. The method of claim 49, wherein the antibody isadministered to the subject intravenously and subcutaneously.
 53. Themethod of claim 50, wherein the antibody is administered to the subjectintravenously and subcutaneously.
 54. The method of claim 51, whereinthe antibody is administered in combination with a second therapeuticagent.
 55. The method of claim 52, wherein the antibody is administeredin combination with a second therapeutic agent.
 56. The method of claim53, wherein the antibody is administered in combination with a secondtherapeutic agent.